Monolithic Stationary Phases Research Articles

Professor Carla Bottoli, an enthusiastic scientific researcher, kindly spoke to BrJAC

Carla Beatriz Grespan Bottoli holds a degree in Industrial Chemistry from the Federal University of Santa Maria (1996), as well as a Ph.D. in Chemistry (2002), and a postdoctoral degree in Chemistry (2002-2004), both from the State University of Campinas (Unicamp). In 2005, she undertook an internship at the Institute of Nuclear Sciences of the National Autonomous University of Mexico (UNAM) to study radiolysis in vitamins using gamma radiation from a source of Cobalt 60. In 2017, she presented her habilitation thesis (associate professor's thesis) at Unicamp. Currently, she is Associate Professor and Postgraduate Advisor at the Institute of Chemistry at Unicamp (IQ-Unicamp), where she works in the area of analytical chemistry, with emphasis on separation methods, mainly liquid chromatography. Prof. Bottoli coordinates the NovaCrom – Liquid Chromatography Laboratory (https://novacromlab.wixsite.com/novacrom), which is a research group that has been working on the development of new monolithic stationary phases for capillary liquid chromatography. Research is also focused on the development of analytical methods that employ liquid chromatography coupled with mass spectrometry to study the translocation of pesticides in plants such as coconut, palm, and soybean. Between 2019 and 2021, Prof. Bottoli was an associate coordinator of the Research Commission of the IQ-Unicamp and is currently a member of the Research Integrity Commission at Unicamp. She is a member of the Brazilian Chemical Society and a researcher at the National Institute of Science and Technology in Bioanalytics (INCTBio). Carla Bottoli is the mother of Murilo, 13 and Mateus, 10.

A novel chiral monolithic nano-column with 50 µm i.d. for the enantioseparation of chiral drugs by nano-liquid chromatography

A novel chiral monolithic nano-column was developed for the enantioseparation of chiral drugs by nano-liquid chromatography (nano-LC). The monolithic capillary with 50 µm i.d. was first prepared by an in-situ polymerization using butyl methacrylate (BuMA), ethylene dimethacrylate (EDMA) and NFMoc-Lysine modified GO nanoparticles. The final monolithic stationary phase was characterized by using FT-IR spectra of synthesized structures, scanning electron microscopy (SEM) and chromatographic analyses. After characterization, the prepared chiral monolithic column was investigated for the chiral separation of pharmaceutical racemates, including anti-inflammatory drugs (e.g., ketoprofen, ibuprofen, naproxen, etodolac and flurbiprofen) and β-blockers (e.g. propranolol, metoprolol and atenolol). It was demonstrated that NFMoc-Lysine modified GO nanoparticles incorporated chiral monolith could be successfully developed while this chiral monolith allowed sensitive chiral separation of chiral drugs. The chiral separation could be mostly achieved via the formation of π-π interactions as well as hydrophilic interactions between chiral stationary phase and enantiomers

Monolithic stationary phases prepared via cyclic anhydride ring-opening polymerization as tunable platforms for chromatographic applications.

Polymer monolithic stationary phases were prepared based on a cyclic anhydride as a reactive and tunable platform via ring-opening post-polymerization using primary amines, octadecylamine and benzylamine. The characterization techniques indicated the insertion of the functional groups into the original monoliths and confirmed the amidation reactions. The post-polymerization modification also improved the monolith's thermal and mechanical stability and induced significant improvement in their surface area. The stationary phases were synthesized inside small dimension stainless-steel columns (2.1 mm i.d. × 50 mm length). The prepared columns before and after modifications have been tested for the separation of the alkylbenzene series and some polycyclic aromatic hydrocarbons (PAHs) as model compounds. In all cases, the chromatographic performance in terms of the height equivalent to a theoretical plate on the functionalized monoliths was remarkably improved when compared with that on the unmodified monolith, which was between 9.59-39.49 μm and 4.08-31.50 μm using monoliths modified with octadecylamine and benzylamine, respectively. Under the same chromatographic conditions, the functionalization of monoliths with octadecylamine provided more hydrophobic interactions and enhanced the retention of alkylbenzenes, while the modification of monoliths with benzyl groups improved the separation and the retention of the PAHs through the strong π-π interactions. However, post-modification polymerization with octadecylamine and benzylamine enhanced the separation efficiency of the prepared columns toward all studied compounds. The repeatability of the injections on the same column and the reproducibility of the prepared columns have been studied for some selected parameters and estimated in terms of percent relative standard deviation (%RSD) for some of the studied compounds. The repeatability of the prepared columns was ≤9.42% (n = 5) based on run-to-run injections and ≤9.48% based on day-to-day injections for five successive days. The reproducibility levels, on the other hand, were ≤20.95% for all studied parameters in all cases. To assess their performance for the analysis of real samples, the applicability of the prepared columns was examined for the separation of the active ingredients extracted from some commercial pharmaceutical formulations and for the separation of tea water extract constituents. The validation data show the suitability of the columns for practical use in the routine analysis of these samples.

HPLC method for simultaneous quantification of lumacaftor and ivacaftor bulk and pharmaceutical formulations

In 2015, the Food and Drug Administration granted approval for the use of lumacaftor 200 mg and ivacaftor 125 mg in the treatment of cystic fibrosis patients who possess the F508del mutation, namely those who are 12 years of age or older. Since its approval, the medicine has been implemented in clinical settings, although the presence of numerous disputes, with the aim of mitigating disease symptoms and enhancing the overall quality of life. Given the existing gaps in the literature regarding the analysis of the amalgamation of these two active substances, a straightforward and practical HPLC approach has been devised in adherence to the guidelines outlined in the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q2(R1) document. To accomplish this objective, the process of separation was successfully carried out using a monolithic silica stationary phase (Chromolith High Resolution RP-18e, 100 mm × 4.6 mm i.d., Merck KGaA, Darmstadt, Germany). The separation process was conducted using a gradient mode. The initial composition of the mobile phase consisted of acetonitrile and a phosphate buffer solution with a concentration of 0.030 M and a pH of 3.5. The flow rate was recorded as 1.0 mL/min, and avanafil was used as an internal standard. The improved and verified approach has demonstrated successful application in bulk and pharmaceutical formulation evaluations when utilizing the ivacaftor/lumacaftor combination.

Selective immunoglobulin aggregates removal in antivenoms by a simple chromatographic step based on a monolithic stationary phase

Antivenom therapy is a critical intervention for treating the more than 5.000.000 envenomation accidents that occur each year around the world. These immunotherapeutic drugs are mostly produced following techniques developed more than fifty years ago with minor changes. Aggregate content has been described as one of the main causes of early adverse effects after intravenous administration of antivenoms. In this work we propose the introduction of a final polishing step to traditional antivenom manufacturing processes aimed at lowering the aggregate content in the final product. The refinement step proposed in this work is based on the selective capture of immunoglobulin aggregates by a cation exchange monolithic stationary phase. We show that this media can effectively remove aggregates in the final product under isotonic ion-strength and mildly acidic conditions following a negative chromatography strategy, thus making it a useful technique for producing higher quality products.

Polysaccharide and Cyclodextrin-Based Monolithic Chiral Stationary Phases and its Application to Chiral Separation.

The recent development of monolithic chiral stationary phases (CSPs) for liquid chromatography (LC) is mainly focused on reducing backpressure, maximizing flow rates, faster run time, column efficiency, and stability. This review paper emphasizes recent progress in the development of polysaccharide and cyclodextrin-based monolithic CSPs. Further the paper draws attention to competing techniques, like non-porous particle-packed columns, core-shell and monoliths as chromatographic support matrix, available for achieving fast and efficient chromatographic separation. A brief discussion on the three main classes of chiral monolithic stationary phase viz. silica, organic polymer and hybrid-based monolithic stationary phases is also presented. In addition, the paper highlights various studies on the application of monolith chiral CSPs in LC and capillary electrochromrography separation and analysis of chiral compounds.

Characterization of hybrid organo-silica monoliths for possible application in the gradient elution of peptides.

We characterized thermally polymerized organo-silica hybrid monolithic capillaries to test their applicability in the gradient elution of peptides. We have used a single-pot approach utilizing 3-(methacryloyloxy)propyltrimethoxysilane (MPTMS), ethylene dimethacrylate (EDMA), and n-octadecyl methacrylate (ODM) as functional monomers. The organo-silica monolith containing MPTMS and EDMA was compared with the stationary phase prepared by adding ODM to the original polymerization mixture. Column prepared using a three-monomer system provided a lower accessible volume of flow-through pores, a higher proportion of mesopores, and higher efficiency. We utilized isocratic and gradient elution data to predict peak widths in gradient elution. Both protocols provided comparable results and can be used for peptide peak width prediction. However, applying gradient elution data for peak width prediction seems simpler. Finally, we tested the effect of gradient time on achievable peak capacity in the gradient elution of peptides with a column prepared with a three-monomer system providing a higher peak capacity. However, the performance of hybrid organo-silica monolithic stationary phases in gradient elution of peptides must be improved compared to other monolithic stationary phases. The limiting factor is column efficiency in highly aqueous mobile phases, which needs to be focused on.

Research progress on preparation and applications of covalent organic framework-based chromatographic stationary phases

Given continuous developments in industrial and scientific research, the separation and analysis of complex systems with high sensitivity, throughput, and selectivity is facing new challenges. Chromatography plays an irreplaceable role in separation science and is widely applied in environmental monitoring, pharmaceutical analysis, and food safety. Owing to their outstanding advantages, such as high loading capacity, precise quantification, and good reproducibility, chromatographic separation techniques based on various retention mechanisms have been utilized to detect different analytes. The stationary phase is the core material of chromatographic columns and has an extremely important influence on their separation performance. The selectivity and efficiency of separation largely depend on the chromatographic stationary phase. However, traditional stationary phases, such as silicon-based matrices, are characterized by complex preparation processes, poor permeability, large mass transfer resistance, and a narrow pH range. In addition, polymer matrices show poor mechanical stability and susceptibility to swelling, which limit their applications in the field of separation. Therefore, the development of novel stationary phases with the advantages of traditional stationary phases has become a research emphasis in the field of analytical science in efforts to meet separation requirements under different environments. Various stationary phases based on novel porous materials, such as metal organic frameworks (MOFs), porous organic cages (POCs), and covalent organic frameworks (COFs), are used for chromatographic separation. As mesh crystalline porous materials, MOFs have the advantages of a large surface area, adjustable structure, and easy functionalization; thus, they are widely used as chromatographic stationary phases in reverse-phase chromatography, hydrophilic-mode chromatography, mixed-mode chromatography, and other separation modes. However, because the pore size of MOFs is small and most MOFs demonstrate poor chemical stability under acidic or alkaline conditions, their applications in chromatographic separation are greatly limited. COFs are porous, crystalline polymer materials composed of light elements (H, O, C, N, B, and Si) connected via covalent bonds. Their advantages include a low density, large specific surface area, high porosity, good chemical and thermal stability, regular pores, and adjustable pore sizes. Because of their unique structures and properties, COFs are widely used in many fields such as catalysis, enrichment, gas capture, and sensing. COF materials are also suitable for separation analysis and considered ideal materials for novel chromatographic stationary phases. This review summarizes the latest research progress on the preparation and applications of COF-based chromatographic stationary phases over the past five years. First, the preparation of COF-based stationary phases (SiO2@COFs stationary phase, COFs monolithic stationary phase, pure COFs stationary phase and COFs-coated stationary phase) is introduced. The latest applications of COF-based stationary phases in the separation of organic compounds, isomers, and chiral compounds are then described in detail. Finally, the future development trends and challenges of chromatographic stationary phases based on COFs are discussed to provide new ideas for the future design and development of novel chromatographic stationary phases based on COFs.

A monolithic stationary phase with dendritic nanostructures for the separation of PEGylated proteins.

Separation of PEGylated protein mixtures into individual species is a challenging procedure, and many efforts have been focused on creating novel chromatographic supports for this purpose. In this study, a new monolithic stationary phase with hyperbranched nanostructures was chemically synthesized. For this, monoliths with a support matrix of poly (glycidyl methacrylate-co-ethylene dimethacrylate) and ethylenediamine chemistry were modified with third-generation dendrons with butyl-end groups. The new monolith was analyzed by infrared spectroscopy, confirming the dendron with butyl ligands and exhibited low mass transfer resistance as observed by breakthrough frontal analysis. This support was able to separate mono-PEG ribonuclease A from the PEGylation mixture, indicated by a single band (∼30kDa) in the electrophoretic analysis. Moreover, the separation of mono-PEGylated positional isomers was probably observed, as the protein with ∼30kDa was found in two separate peaks. Interestingly, the dendronized monolith allowed the separation of the reaction mixture into individual PEGylated species when using high ammonium sulfate concentrations (2M). A correlation between the PEGylation degree and the strength of the hydrophobic interactions on the monolith was observed. This chromatographic approach combines the natural branched architecture of dendrons and the higher capabilities of the monoliths enhancing the hydrophobic surface area, and therefore the interaction between the PEGylated proteins and ligands. Thus, the novel support represents a novel platform for the purification of PEGylated from non-PEGylated proteins with biotechnological applications.

A protocol for fabrication of polymer monolithic capillary columns and tuning the morphology targeting high-resolution bioanalysis in gradient-elution liquid chromatography.

Polymer monolithic stationary phases are designed as a continuous interconnected globular material perfused by macropores. Like packed column, where separation efficiency is related to particle diameter, the efficiency of monoliths can be enhanced by tuning the size of both the microglobules and macropores. This protocol described the synthesis of poly(styrene-co-divinylbenzene) monolithic stationary phases in capillary column formats. Moreover, guidelines are provided to tune the macropore structure targeting high-throughput and high-resolution monolith chromatography. The versatility of these columns is exemplified by their ability to separate tryptic digests, intact proteins, and oligonucleotides under a variety of chromatographic conditions. The repeatability of the presented column fabrication process is demonstrated by the successful creation of 12 columns in three different column batches, as evidenced by the consistency of retention times (coefficients of variance [c.v.]=0.9%), peak widths (c.v.=4.7%), and column pressures (c.v.=3.1%) across the batches.

Ni(II) functionalized polyhedral oligomeric silsesquioxane based capillary monolith for purification of histidine-tagged proteins by immobilized metal affinity micro-chromatography

A new capillary monolithic stationary phase was synthesized for the purification of histidine tagged proteins by immobilized metal affinity micro-chromatography (μ-IMAC). For this purpose, mercaptosuccinic acid (MSA) linked-polyhedral oligomeric silsesquioxane [MSA@poly(POSS-MA)] monolith 300 μm in diameter was obtained by thiol-methacrylate polymerization using methacryl substituted-polyhedral oligomeric silsesquioxane (POSS-MA) and MSA as the thiol functionalized agent in a fused silica capillary tubing. Ni(II) cations were immobilized onto the porous monolith via metal-chelate complex formation with double carboxyl functionality of bound MSA segments. μ-IMAC separations aiming the purification of histidine tagged-green fluorescent protein (His-GFP) from Escherichia coli extract were carried out on Ni(II)@MSA functionalized-poly(POSS-MA) [Ni(II)@MSA@poly(POSS-MA)] capillary monolith. His-GFP was succesfully isolated by μ-IMAC on Ni(II)@MSA@poly(POSS-MA) capillary monolith with the isolation yield of 85 % and the purity of 92 % from E. coli extract. Higher His-GFP isolation yields were obtained with lower His-GFP feed concentrations and lower feed flow rates. The monolith was used for consecutive His-GFP purifications with a tolerable decrease in equilibrium His-GFP adsorption over five runs.

Nano-liquid chromatography with monolithic stationary phase based on naphthyl monomer for proteomics analysis

Monolithic poly(2-vinylnaphthalene-co-divinylbenzene) columns were introduced, for the first time, and were evaluated as the separation media for nano-liquid chromatography (nano-LC). These columns were prepared by in-situ polymerization of 2-vinylnaphthalene (2-VNA) as the functional monomer and divinylbenzene (DVB) as the crosslinker in a fused silica capillary column of 50 µm i.d. Various porogenic solvents, including tetrahydrofuran (THF), dodecanol and toluene were used for morphology optimization. Final monolithic column (referred to as VNA column) was characterized by using scanning electron microscopy (SEM) and chromatographic analyses. Alkylbenzenes (ABs), and polyaromatic hydrocarbons (PAHs) were separated using the VNA column while the column offered excellent hydrophobic and π-π interactions under reversed-phase conditions. Theoretical plates number up to 41,200 plates/m in isocratic mode for ethylbenzene could be achieved. The potential of the final VNA column was demonstrated with a gradient elution in the separation of six intact proteins, including ribonuclease A (RNase A), cytochrome C (Cyt C), lysozyme (Lys), β-lactoglobulin (β-lac), myoglobin (My) and α-chymotrypsinogen (α-chym) in nano LC system. The column was then applied to the peptide analysis of trypsin digested cytochrome C, allowing a high peak capacity up to 1440 and the further proteomics analysis of COS-7 cell line was attempted applying the final monolithic column in nano-LC UV system.

An Overview of Monolithic Column: Types, Parameters and Applications

The column is the main component for chromatographic separation. Nowadays, monolithic columns are graining more popularity in the field of separation media for liquid chromatography. The monolith columns possess great potential as compared to the conventional packed column in terms of preparing complex mixtures. These columns provide various properties like higher permeability, high-efficiency fast separations, high flow rate with lower backpressure, fast mass transfer kinetics with a high binding capacity. It is categories into three columns and they are organic monolithic column, inorganic monolithic column and hybrid monolithic column and all three types of monolithic column differ through their porous properties. In this review, the various advantage of the high-efficiency monolithic column with recent advances, the origin of the concept, the various parameter of the monolithic stationary phase and the application of monolithic columns are illustrated. It is better column in comparison of selectivity, reproducibility and performance.
 Keywords: Monolithic column, Packed columns, Inorganic and organic monolithic column, Column parameters, Pharmaceutical Applications

Rapid fabrication of zwitterionic sulfobetaine vinylimidazole-based monoliths via photoinitiated copolymerization for hydrophilic interaction chromatography

Zwitterionic sulfobetaine-based monolithic stationary phases have attracted increasing attention for their use in hydrophilic interaction chromatography. In this study, a novel hydrophilic polymeric monolith was fabricated through photo-initiated copolymerization of 3-(3-vinyl-1-imidazolio)-1-propanesulfonate (SBVI) with pentaerythritol triacrylate using methanol and tetrahydrofuran as the porogenic system. Notably, the duration for the preparation of this novel monolith was as little as 5 min, which was significantly shorter than that required for previously reported sulfobetaine-based monoliths prepared via conventional thermally initiated copolymerization. Moreover, these monoliths showed good morphology, permeability, porosity (62.4%), mechanical strength (over 15 MPa), column efficiency (51,230 plates/m), and reproducibility (relative standard deviations for all analytes were lower than 4.6%). Mechanistic studies indicated that strong hydrophilic and negative electrostatic interactions might be responsible for the retention of polar analytes on the zwitterionic SBVI-based monolith. In particular, the resulting monolith exhibited good anti-protein adhesion ability and low nonspecific protein adsorption. These excellent features seem to favor its application in bioanalysis. Therefore, the novel zwitterionic sulfobetaine-based monolith was successfully employed for the highly selective separation of small bioactive compounds and the efficient enrichment of N-glycopeptides from complex samples. In this study, we prepared a novel zwitterionic sulfobetaine-based monolith with good performance and developed a simpler and faster method for preparation of zwitterionic monoliths.

Current trends in the development of polymer-based monolithic stationary phases.

This review focuses on the development and applications of organic polymer monoliths, with special attention to the literature published in 2021. The latest protocols in the preparation of polymer monoliths are discussed. In particular, tailored surface modification using nanomaterials, the development of chiral stationary phases and development of stationary phases for capillary electrochromatography are reviewed. Furthermore, the optimization of pore forming solvents composition is also discussed. Finally, the use of monolithic stationary phases in sample treatment using solid-phase extraction and enrichment methods, molecularly imprinted polymers and enzymatic reactors is mentioned.

Cobalt (II)-Mediated Molecularly Imprinted Polymer as a Monolithic Stationary Phase for Separation of Racemic Citronellal by Liquid Chromatography.

A metal-mediated molecularly imprinted polymer (MMIP) monolithic column was prepared as the stationary phase for high-performance liquid chromatography (HPLC) and applied to the enantiomeric separation of rac-citronellal. MMIP column was prepared through in situ copolymerizations with the ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate/[BMIM][BF4] as the primary pore-forming agent and cobalt(II) acetate as the metal pivot. Interactions between polymer components in the synthesized monolith were assessed by FTIR to identify the functional groups. The monolith morphology was characterized with SEM, and the template removal was detected by UV Spectrophotometry at 253 nm. In this study, (R)-(+)-citronellal was used as a template, whereas 4-vinylpyridine (4-VP) was employed as the functional monomer with two monomer crosslinkers, trimethylolpropane trimethacrylate (TRIM), and ethylene glycol dimethacrylate (EDMA). The ternary mixture of porogenic solvent consisted of [BMIM][BF4], dimethylformamide (DMF), and dimethyl sulfoxide (DMSO) with the applied ratio of 1 : 1:1 (v/v) and 10 : 1:5 (v/v) for the preparation of MMIP using TRIM and EDMA crosslinkers, respectively. Co(II) ion was added to the porogenic solvent before being mixed with the functional monomer and the crosslinker mixtures. Separating the rac-citronellal was achieved on the synthesized MMIP, showing better selectivity than the monolithic metal-mediated nonimprinted polymer (MNIP), nonimprinted polymer (NIP), and molecularly imprinted polymer (MIP).

Characterization and applications of a trioctyl(3/4-vinylbenzyl)phosphonium stationary phase for use in capillary liquid chromatography

The morphology, composition, and selectivity of a silica-based monolithic stationary phase, grafted by a layer of trioctyl(3/4-vinylbenzyl)phosphonium chloride ([P888VBn]Cl), is presented. The results of elemental analysis confirmed that the prepared stationary phase contains 38.8 at.% of silicon, 60.2 at.% of carbon, and 1.0 at.% of phosphorus. Capillary columns (150 × 0.1 mm) for liquid chromatography were evaluated using alkylbenzenes, monosubstituted benzenes, polyaromatic compounds, substituted benzene regioisomers, and aromatic carboxylic and amino acids. The prepared ionic liquid (IL)-based stationary phase exhibits hydrophobic, hydrophilic, and electrostatic interactions, as confirmed by experiments on the evaluation of the effect of the mobile phase composition (content of acetonitrile and ammonium formate) on the isocratic chromatographic separation. Thus, the IL-based capillary column demonstrates a unique separation selectivity compared to Phenyl-, C8-, and C18-stationary phases, and high efficiency for an expanding number of structurally diverse compounds.

Enantioseparation by simultaneous biphasic recognition using mobile phase additive and chiral stationary phase in capillary electrochromatography

Biphasic chiral recognition was first applied for the enantioseparation acidic drugs in capillary electrochromatography. The biphasic recognition system was composed of mobile phase additive (β-cyclodextrin) and monolithic chiral stationary phase (proteins). The pretreated silica-fused capillary was treated with 3-trimethoxysilyl propyl methacrylate to attach double bond ligand onto the surface. The mixed monolithic chiral stationary phase was constructed with bovine serum albumin and pepsin using one-pot polymerization by chemical covalent coupling, while dimethyl sulfoxide and methanol were used as the progenic solvents. The effects of the type and concentration of β-cyclodextrin additive as well as pH value of the mobile phase on the separation efficiency were optimized. The performance of the biphasic recognition system was validated by separating acidic drugs (such as ketoprofen, ibuprofen, loxoprofen, flurbiprofen and carprofen) in capillary electrochromatography, achieving outstanding separation efficiency. In terms of migration time and resolution, the run-to-run, intra-day, and inter-day repeatability through relative standards deviation were within 5.0%, exhibiting excellent stability of the biphasic recognition system. Conceivably, the experimental result reveals that biphasic chiral recognition capillary electrochromatography offers a promising prospect for enantioseparation of chiral compounds in a highly efficient manner.

Poly(acrylamide-co-N,N'-methylenebisacrylamide) Monoliths for High-Peak-Capacity Hydrophilic-Interaction Chromatography-High-Resolution Mass Spectrometry of Intact Proteins at Low Trifluoroacetic Acid Content.

In this study, we optimized a polymerization mixture to synthesize poly(acrylamide-co-N,N′-methylenebisacrylamide) monolithic stationary phases for hydrophilic-interaction chromatography (HILIC) of intact proteins. Thermal polymerization was performed, and the effects of varying the amount of cross-linker and the porogen composition on the separation performance of the resulting columns were studied. The homogeneity of the structure and the different porosities were examined through scanning electron microscopy (SEM). Further characterization of the monolithic structure revealed a permeable (Kf between 2.5 × 10–15 and 1.40 × 10–13 m2) and polar stationary phase suitable for HILIC. The HILIC separation performance of the different columns was assessed using gradient separation of a sample containing four intact proteins, with the best performing stationary phase exhibiting a peak capacity of 51 in a gradient of 25 min. Polyacrylamide-based materials were compared with a silica-based particulate amide phase (2.7 μm core–shell particles). The monolith has no residual silanol sites and, therefore, fewer sites for ion-exchange interactions with proteins. Thus, it required lower concentrations of ion-pair reagent in HILIC of intact proteins. When using 0.1% of trifluoroacetic acid (TFA), the peak capacities of the two columns were similar (30 and 34 for the monolithic and packed column, respectively). However, when decreasing the concentration of TFA to 0.005%, the monolithic column maintained similar separation performance and selectivity (peak capacity 23), whereas the packed column showed greatly reduced performance (peak capacity 12), lower selectivity, and inability to elute all four reference proteins. Finally, using a mobile phase containing 0.1% formic acid and 0.005% TFA, the HILIC separation on the monolithic column was successfully hyphenated with high-resolution mass spectrometry. Detection sensitivity for protein and glycoproteins was increased and the amount of adducts formed was decreased in comparison with separations performed at 0.1% TFA.

Chromatographic method for the determination of inflammatory biomarkers and uric acid in human saliva

Determination of concentration of biomarkers of the activation of immune system, uric acid, and creatinine in the saliva can be useful tool for the diagnosis and monitoring of early manifestations of diseases such as malignant, inflammatory, and periodontal disorders. We have developed and validated a high-performance liquid chromatographic method coupled with fluorescence and diode array detection for the separation and quantification of neopterin, tryptophan, creatinine, uric acid, and kynurenine in the human saliva. A separation of these analytes was achieved within 9 min by using second-generation monolithic stationary phase and elution with phosphate buffer. The present method involves very simple sample preparation requiring small amount of sample matrix. The internal standard 3-nitro-l-tyrosine was used for a more precise quantification. The sensitivity of the present method was demonstrated with lower limits of quantification of 0.6 × 10−3 μmol/L for neopterin, 0.725 μmol/L for tryptophan, 0.12 μmol/L for creatinine, 0.18 μmol/L for uric acid, and 0.135 μmol/L for kynurenine. The method was validated with 67 real-life saliva samples collected from patients suffering from breast, ovarian, colorectal, and renal cancer, and 19 saliva samples from patients with periodontal diseases and allowed monitoring of inflammatory response.