Longevity and other practical benefits of monolithic silica columns in the analysis of samples with complex matrices.

At the turn of the millennium, the monolithic columns invoked new chances in HPLC. Even more than their organic polymer-based siblings, the inorganic silica-based monoliths targeted the territory of classical fully porous particle-packed columns, promising many benefits. Based on the number of published articles, the monoliths attracted academics just in the first few years after their introduction to the market. Lately, as superficially porous particles and sub-2-micron fully porous particles dominated the market, they stayed in the focus of routine laboratories and those who really appreciated the high porosity of the monolithic bed. The monoliths' practical benefits cannot be easily traced in the literature when they gradually lose academics' interest. Nevertheless, after more than 20 years of our experience, we still favor silica monoliths for their low back pressure and longevity when analyzing samples of clinical, pharmaceutical, and environmental origin. At the same time, the high permeability of monoliths enabled the birth of sequential injection chromatography, the medium-pressure separation technique based on the flexible flow manifold. This minireview aims to check, discuss, and summarize the practical aspects of monolithic silica columns in HPLC and medium-pressure sequential injection chromatography (SIC) that may not be visible at first sight but are evident retrospectively.

Design of a Biocatalytic Flow Reactor Based on Hierarchically Structured Monolithic Silica for Producing Galactooligosaccharides (GOSs).

Climate change mitigation requires the development of greener chemical processes. In this context, biocatalysis is a pivotal key enabling technology. The advantages of biocatalysis include lower energy consumption levels, reduced hazardous waste production and safer processes. The possibility to carry out biocatalytic reactions under flow conditions provides the additional advantage to retain the biocatalyst and to reduce costly downstream processes. Herein, we report a method to produce galactooligosaccharides (GOSs) from a largely available feedstock (i.e. lactose from dairy production) using a flow reactor based on hierarchically structured monolithic silica. This reactor allows for fast and efficient biotransformation reaction in flow conditions.

Phenyl-bonded monolithic silica capillary column liquid chromatographic separation and detection of fluorogenic derivatized intact proteins.

Prior to the identification of proteins for proteomics analysis in human cells, separation of fluorogenic derivatized proteins with a fluorogenic reagent, 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide, has typically been performed by using a conventional reversed-phase HPLC column. However, the number of proteins in human cells (HepaRG) that are separated by this conventional approach is limited to approximately 500. In this study, a nanoflow liquid chromatography system with an evaluated phenyl-bonded monolithic silica capillary column (0.1 mm i.d., 700 mm length) was used to increase the number of separated fluorogenic derivatized proteins. This system was used to separate derivatized human cell proteins (K562) and yeast (Saccharomyces cerevisiae) proteins as model cell proteomes. More than 1,300 protein peaks were separated/detected from both cell proteomes. We present a straightforward comparison of multiple separation profiles using a novel chromatogram display approach, termed the "spiderweb" chromatogram. In addition, to validate that the detected peaks are derived from proteins, a mass spectrometer was connected to the capillary column and deconvolution of the obtained mass spectra was performed. Furthermore, different molecular weight distribution profiles of the expressed proteins were observed between the two cell proteomes.

Chiral Monolithic Silica-Based HPLC Columns for Enantiomeric Separation and Determination: Functionalization of Chiral Selector and Recognition of Selector-Selectand Interaction.

This review draws attention to the use of chiral monolithic silica HPLC columns for the enantiomeric separation and determination of chiral compounds. Properties and advantages of monolithic silica HPLC columns are also highlighted in comparison to conventional particle-packed, fused-core, and sub-2-µm HPLC columns. Nano-LC capillary monolithic silica columns as well as polymeric-based and hybrid-based monolithic columns are also demonstrated to show good enantioresolution abilities. Methods for introducing the chiral selector into the monolithic silica column in the form of mobile phase additive, by encapsulation and surface coating, or by covalent functionalization are described. The application of molecular modeling methods to elucidate the selector-selectand interaction is discussed. An application for enantiomeric impurity determination is also considered.

Development of Validated and Stability-Indicating LC-DAD and LC-MS/MS Methods for Determination of Avanafil in Pharmaceutical Preparations and Identification of a Novel Degradation Product by LCMS-IT-TOF.

Avanafil (AVA), one of the most effective drugs prescribed for erectile dysfunction, is a pyrimidine-derivative PDE5 inhibitor. In the current work, new LC methods were developed and validated for quantitative determination of avanafil and qualitative determination of its degradation products. The quantitative determination of avanafil was carried out using liquid chromatography with photodiode array detection (LC-DAD) and liquid chromatography-tandem mass spectrometry LC-MS/MS methods, and fully validated according to the ICH Q2 (R1) guideline, while qualitative determination was performed using a liquid chromatography mass spectrometry-ion trap-time of flight (LCMS-IT-TOF) instrument. The separation of avanafil and its degradation products was carried out using the same reversed-phase chromatographic conditions, in which a second-generation C18-bonded monolithic silica column (Chromolith® High Resolution RP-18e, 100 × 4.6 mm, Merck KGaA) was used as stationary phase. Briefly, the methods enable quantitation of avanafil with high accuracy (recovery > 95%) and precision (RSD% < 2.0), within the ranges of 0.5⁻20 µg/mL for LC-DAD and 150⁻6000 ng/mL for LC-MS/MS. In the forced degradation studies, over and above currently existing data, a new oxidation-based degradation product, whose predicted m/z is 367.1168, was identified and its structure was confirmed by high-resolution mass spectrometric analysis. As the main advantage, either an LC-DAD or LC-MS/MS instrument can be chosen for interference-free quantitation of AVA, according to the facilities in quality-control laboratories.

High-performance liquid chromatography separation of unsaturated organic compounds by a monolithic silica column embedded with silver nanoparticles.

The optimization of a porous structure to ensure good separation performances is always a significant issue in high-performance liquid chromatography column design. Recently we reported the homogeneous embedment of Ag nanoparticles in periodic mesoporous silica monolith and the application of such Ag nanoparticles embedded silica monolith for the high-performance liquid chromatography separation of polyaromatic hydrocarbons. However, the separation performance remains to be improved and the retention mechanism as compared with the Ag ion high-performance liquid chromatography technique still needs to be clarified. In this research, Ag nanoparticles were introduced into a macro/mesoporous silica monolith with optimized pore parameters for high-performance liquid chromatography separations. Baseline separation of benzene, naphthalene, anthracene, and pyrene was achieved with the theoretical plate number for analyte naphthalene as 36,000 m(-1). Its separation function was further extended to cis/trans isomers of aromatic compounds where cis/trans stilbenes were chosen as a benchmark. Good separation of cis/trans-stilbene with separation factor as 7 and theoretical plate number as 76,000 m(-1) for cis-stilbene was obtained. The trans isomer, however, is retained more strongly, which contradicts the long- established retention rule of Ag ion chromatography. Such behavior of Ag nanoparticles embedded in a silica column can be attributed to the differences in the molecular geometric configuration of cis/trans stilbenes.

Hydrothermal preparation of hybrid carbon/silica monolithic capillary column for liquid chromatography.

A simple, easy and economical approach for the preparation of a hybrid carbon/silica monolithic capillary column was described for the first time by using silica monolith as framework in combination with hydrothermal carbonization at 180°C. During the preparation process, formamide was introduced to the reaction solutions to reduce the dissolution rate of monolithic silica skeleton and its optimal concentration was 1.5 M. Fourier transform infrared spectrometry, scanning electron microscopy, energy dispersive X-ray spectrometry, and inverse size exclusion chromatography were carried out to characterize the as-prepared column. The results demonstrated that carbon spheres ranging from 150 to 1000 nm were successfully attached to the surface of silica skeleton. The prepared hybrid carbon/silica column had a permeability of 4.4 × 10(-14) m(2). Chromatographic performance of the column was evaluated by separation of various compounds including alkylbenzenes, nucleosides and bases, and aromatic acids. The column exhibited an efficiency of 75,000 plates/m for butylbenzene at the optimal linear velocity of 0.23 mm/s. The successful separation of these compounds and the study on mechanism indicated that the column can be applied in mixed-mode chromatography.

Simultaneous determination of N(G)-monomethyl-L-arginine, N(G),N(G)-dimethyl-L-arginine, N(G),N(G')-dimethyl-L-arginine, and L-arginine using monolithic silica disk-packed spin columns and a monolithic silica column.

We have developed and validated a high-performance liquid chromatography method that uses monolithic silica disk-packed spin columns and a monolithic silica column for the simultaneous determination of N(G)-monomethyl-L-arginine, N(G),N(G)-dimethyl-L-arginine, and N(G),N(G')-dimethyl-L-arginine in human plasma. For solid-phase extraction, our method employs a centrifugal spin column packed with monolithic silica bonded to propyl benzenesulfonic acid as a cation exchanger. After pretreatment, the methylated arginines are converted to fluorescent derivatives with 4-fluoro-7-nitro-2,1,3-benzoxadiazole, and then the derivatives are separated on a monolithic silica column. L-arginine concentration was also determined in diluted samples. Standard calibration curves revealed that the assay was linear in the concentration range 0.2-1.0 µM for methylated arginines and 40-200 µM for L-arginine. Linear regression of the calibration curve yielded equations with correlation coefficients of 0.999 (r(2)). The sensitivity was satisfactory, with a limit of detection ranging from 3.75 to 9.0 fmol for all four compounds. The RSDs were 4.3-4.8% (intraday) and 3.0-6.8% (interday). When this method was applied to samples from six healthy donors, the detected concentrations of N(G)-monomethyl-L-arginine, N(G),N(G)-dimethyl-L-arginine, N(G),N(G')-dimethyl-L-arginine and L-arginine were 0.05 ± 0.01, 0.41 ± 0.07, 0.59 ± 0.11, and 83.8 ± 30.43 µM (n = 6), respectively.

Tannic Acid as a Versatile Template for Silica Monoliths Engineering with Catalytic Gold and Silver Nanoparticles.

Tannic acid in alkaline solutions in which sol-gel synthesis is usually performed with tetraethoxysilane is susceptible to various modifications, including formation of reactive radicals, oxidation under the action of atmospheric oxygen, self-association, and self-polymerization. Here, a precursor with ethylene glycol residues instead of ethanol was used, which made it possible to synthesize bionanocomposites of tannic acid and silica in one stage in neutral media under normal conditions without the addition of acid/alkali and organic solvents. Silica was fabricated in the form of optically transparent monoliths of various shapes with 2-4 nm pores, the radius of which well correlated with the size of a tannic acid macromolecule in a non-aggregated state. Polyphenol, which was remained in pores of silica matrix, served then as reducing agent to synthesize in situ gold and silver nanoparticles. As shown, these Au@SiO2 and Ag@SiO2 nanocomposites possessed localized surface plasmon resonance and high catalytic activity.

HPLC determination of cefprozil in tablets using monolithic and C18 silica columns.

Cefprozil (CPZ) is a second-generation semi-synthetic cephalosporin antibiotic that commonly exists as the mixture of Z and E diastereoisomers, at the ratio of approximately 9:1. A novel reversed-phase HPLC method for the determination of CPZ in tablets was described. The separation of CPZ diastereoisomers and caffeine (internal standard) was carried out by applying the same analytical and instrumental conditions on two stationary phases, which have different surface chemistries. The columns used in the study were monolithic silica Merck Chromolith Performance RP-18e and conventional C18 silica Phenomenex Synergi Hydro RP columns. In total, 10 µL aliquots of samples were injected into the system and eluted using water-acetonitrile (90:10, v/v) solution, which was pumped through the column at a flow rate of 1.0 mL/min. The analyte peaks were detected at 200 nm using diode array detector with high specificity. CPZ diastereoisomers and caffeine were measured within 13 min using the C18 column, whereas <5 min was required for the monolithic one. Validation studies were performed according to official recommendations. Value of a monolithic column for the assay of diastereoisomers in pharmaceutical tablets was evaluated for the first time and found as a powerful alternative to highly efficient C18 columns.

Performance of functionalized monolithic silica capillary columns with different mesopore sizes using radical polymerization of octadecyl methacrylate.

We report on the possibility to enhance the phase ratio and retention factor in silica monoliths. According to pioneering work done by Núñez et al. [1], this enhancement is pursued by applying a stationary phase layer via radical polymerization with octadecyl methacrylate (ODM) as an alternative to the customary octadecylsilylation (C18-derivatization). The difference in band broadening, retention factor and separation selectivity between both approaches was compared. Different hydrothermal treatment temperatures for the column preparation were applied to produce monolithic silica structures with three different mesopore sizes (resp. 10, 13, and 16 nm, as determined by argon physisorption) while maintaining similar domain size (sum of through-pore and skeleton size). It has been found that the columns with the poly(octadecyl methacrylate)-phase (ODM columns) provided a 60 to 80% higher retention factor in methanol-water mixture compared to the octadecylsilylated (ODS) columns produced by starting from similar silica backbone structures. In acetonitrile-water mixture, the enhancement is smaller (15 to 30% times higher), yet significant. By adjusting the fabrication conditions (for both the preparation of the monolithic backbones and the surface functionalization), the achieved retention factors (up k = 4.89 for pentylbenzene in 80:20% (v/v) methanol/water) are obviously higher than obtained in the pioneering study on ODM monoliths of Núñez et al. [1], and column clogging could be completely avoided. In addition, also separation efficiencies were significantly higher than shown in Ref. [1], with plate heights as low as 5.8 µm. These plate heights are however inferior to those observed on the ODS-modified sister columns. The difference can be explained by the slower intra-skeleton diffusion displayed by the ODM-modified columns, in turn caused by the larger obstruction to diffusion originating from the thicker stationary phase layer.

Rapid Purification of Immunoglobulin G Using a Protein A-​immobilized Monolithic Spin Column with Hydrophilic Polymers.

A rapid purification method was developed for antibody production in Chinese hamster ovary (CHO) cells using a Protein A-immobilized monolithic silica spin column with hydrophilic polymers. Monolithic silica modified with copolymers of 2-hydroxyethylmethacrylate (HEMA) and glycidyl methacrylate (GMA) showed lower non-specific protein absorption than that modified with a silane reagent. The epoxy group of GMA was converted to an amino group, and Protein A was modified by the coupling reagent. The amount of immobilized Protein A was controlled by changing the ratio of GMA to HEMA and the mesopore size of monolith. A modified monolith disk was fixed to a spin column for rapid antibody purification. The linear curves (for the antibody concentrations over 10 - 300 µg/mL) had a correlation coefficient of >0.999. Our column had various analytical advantages over previously reported columns, including a shorter preparation time (<10 min) and smaller sample volumes for purification with Protein A-immobilized agarose.

A Novel and Sensitive LC-MS/MS Method for the Quantitation of Ceftiofur in Pharmaceutical Preparations and Milk Samples.

INTRODUCTION: In the present study, a sensitive and selective liquid chromatographytandem mass spectrometry (LC-MS/MS) method was described for the determination of ceftiofur (CEF) in cow milk and pharmaceutical preparations. CEF is an antibiotic compound, which is commonly used in the treatment of animal diseases such as respiratory system, soft tissue, and foot infections, as well as postpartum acute puerperal metritis. One of the critical features of CEF is its prescription while breastfeeding cows; in accordance, its quantitative estimation is essential to assess its residual amounts. METHODS: In the method reported herein, after simple protein precipitation using acetonitrile, the pre-treated samples were introduced into an LC-MS/MS instrument equipped with a Chromolith® High-Resolution RP-18 series HPLC column (100 mm × 4.6 mm from Merck KGaA, Germany). Electrospray ionization was employed as the ionization source in the triple-quadrupole tandem mass spectrometer. RESULTS: For the calibration method using solvent-based standards, LOQ was 3.038 ng/mL, 12.15 ng/mL, and LOD was 1.215 ng/mL and 6.076 ng/mL for ESI+ and ESI- modes, respectively. On the other hand, for the method of matrix-matched standards, LOQ was 1.701 ng/mL, 10.13 ng/mL, and LOD was 0.486 ng/mL and 5.929 ng/mL for ESI+ and ESI- modes, respectively as obtained from signal to noise ratio. CONCLUSION: Applicability of both positive and negative ion modes was tested, and the analyte was detected via multiple reaction monitoring. The distorting effects of the milk matrix on the MS ionization and quantitation of CEF were overcome by using matrix-matched calibration for the first time.

On the identification and quantification of proton-initiated species in the synthesis of poly(2-alkyl-2-oxazoline)s by high resolution liquid chromatography.

Hydrophilic poly(2-oxazoline)s represent a promising alternative to replace poly(ethylene glycol) in the biomedical field. For that purpose, reliable analytical protocols to confirm identity and quantity of impurities are required. In particular, side products deriving from chain transfer reactions occurring during the cationic ring-opening polymerization and incomplete end-capping processes may be present. The analytical approach must hence be capable of separating polymers according to minor changes regarding their end group. We demonstrate that liquid chromatography, relying on a monolithic C18-modified silica column and isocratic as well as gradient elution using water / acetonitrile mixtures and varying detectors, can accomplish such demanding high resolution separations. Poly(2-ethyl-2-oxazoline)s (PEtOx) with acetyl, hydroxyl, and phthalimide ω-end groups were investigated. Identification of side products was achieved through coupling with electrospray ionization mass spectrometry. UV / Vis detection was applied to quantify chain transfer products in PEtOx comprising biphenyl moieties. In addition, gradient elution enabled the separation of PEtOx into macromolecules according to their specific degrees of polymerization in molar mass ranges around 2,000 g mol-1.

Recent Progress in FD-LC-MS/MS Proteomics Method.

Through the course of our bio-analytical chemistry studies, we developed a novel proteomics analysis method, FD-LC-MS/MS (fluorogenic derivatization-liquid chromatography-tandem mass spectrometry). This method consists of fluorogenic derivatization (FD), LC separation, and detection/quantification of the derivatized proteins, followed by isolation, tryptic digestion of the isolated proteins, and final identification of the isolated proteins using electrospray ionization nano-LC-MS/MS of the generated peptide mixtures with a probability-based protein identification algorithm. In this review, we will present various examples where this method has been used successfully to identify expressed proteins in individual human cells. FD-LC-MS/MS is also suitable for differential proteomics analysis. Here, two biological samples are treated by the same steps mentioned above, and the two chromatograms obtained are compared to identify peaks with different intensities (variation in protein levels). Associated peak fractions are then isolated, and the differentially expressed proteins between the two samples are identified by LC-MS/MS. Several biomarkers for cancers have been identified by FD-LC-MS/MS. For more efficient separation, nano-flow LC with a phenyl-bonded monolithic silica-based capillary column was adopted for cell-expressed intact protein analysis. The derivatized human cell proteins (K562) and yeast cell (Saccharomyces cerevisiae) proteins as model intact cell proteins were analyzed by nano-flow LC with fluorescence detection. More than 1,300 protein peaks were separated/detected from both cells. For straightforward comparison of multiple peak separation profiles, a novel type of chromatogram display, termed the "spiderweb" chromatogram, was developed. A nano-LC-FD-LC-mass spectrometry trial for molecular weight estimation of FD proteins has also been conducted.

Performance of small-domain monolithic silica columns in nano-liquid chromatography and comparison with commercial packed bed columns with 2 µm particles.

We report on a direct comparison of the separation performance in capillary nano-LC between commercial packed bed columns and the small-domain silica monoliths in applications. Octadecylsilylated monolithic silica capillary columns with a 50 and 100 µm inner diameter (i.d.) were prepared with a procedure providing domain sizes in the sub-2 µm range. The fabricated monolith columns could provide plate heights (H) of 4.0‒4.2 µm for hexylbenzene (retention factor (k) = 3.6) at an optimal linear velocity range under an isocratic condition, while showing a column permeability (Kv0 = 1.6‒1.8 × 10-14 m2) comparable to that of a column packed with 3‒3.5 µm particles. When the peak capacity (np) for a cytochrome C digest was compared for variable gradient times (tG = 15, 30, 60, and 120 min) and constant gradient steepness (b'), the present monolith columns could show a 30‒40% higher np-value than the packed capillary column with 2 µm particles (e.g. np = 180 versus np = 259 at tG = 30 min). The produced monolith columns showed a high chromatographic repeatability for both isocratic and gradient elution (e.g. relative standard deviation (n = 3, RSD (%)) = 0.5% for H, 2,6% for k, and 5.6% for Kv0 in the isocratic mode using the 100 µm i.d.-columns). The present results show that the domain sizes which can now be achieved for capillary silica monoliths are sufficiently small to result in separation efficiencies that can successfully compete with the commercial packed bed columns available for use in nano-LC applications.

Development of an integrated chromatographic system for ω-transaminase-IMER characterization useful for flow-chemistry applications.

An integrated chromatographic system was developed to rapidly investigate the biocatalytic properties of ω-transaminases useful for the synthesis of chiral amines. ATA-117, an (R)-selective ω-transaminase was selected as a proof of concept. The enzyme was purified and covalently immobilized on an epoxy monolithic silica support to create an immobilized enzyme reactor (IMER). Reactor efficiency was evaluated in the conversion of a model substrate. The IMER was coupled through a switching valve to an achiral analytical column for separation and quantitation of the transamination products. The best conditions of the transaminase-catalyzed bioconversion were optimized by a design of experiments (DoE) approach. The production of (R)-1-(4-methoxyphenyl)propan-2-amine and (R)-1-methyl-3-phenylpropylamine, intermediates for the synthesis of the bronchodilator formoterol and the antihypertensive dilevalol respectively, was achieved in the presence of different amino donors. The enantiomeric excess (ee) was determined off-line by developing a derivatization procedure using Nα-(2,4-dinitro-5-fluorophenyl)-L-alaninamide reagent. The most satisfactory conversion yields were 60% for (R)-1-(4-methoxyphenyl)propan-2-amine and 29% for (R)-1-methyl-3-phenylpropylamine, using isopropylamine as amino donor. The enantiomeric excess of the reactions were 84%R and 99%R, respectively.

Hyphenation of short monolithic silica capillary column with vacuum ultraviolet spectroscopy detector for light hydrocarbons separation.

Compared to conventionnal bench top instruments, on-line GC analyzers require specific characteristics. On one hand, for some applications operating with a reactor pressure as high as several tens of bars, sample pressure has to be reduced before GC separation, or specific valves and columns have to be designed to perform separation with high carrier gas inlet pressure. On the other hand, informative detectors such as mass spectrometer are valuable but low maintenance detectors are prefered. To fit these two requirements (sampling at high pressure without decompression stage, and informative detector with low maintenance), short monolithic silica capillary column operated with inlet pressure as high as 60 bar has been hyphenated to VUV detector. Injection and column performance have been first investigated. The system has been optimized by adjusting split ratio at high pressure and by tuning two main VUV detector parameters ("average number" linked to data point averaging and make-up gas pressure) to decrease the limit of quantification. The optimization stage led to a set of experimental parameters which is a good compromise between signal-to-noise ratio and chromatographic efficiency. Finally, the hyphenated monolithic column has be used to partially separate a mixture of methane, ethane, carbon monoxide and carbon dioxide within 15 s, and the VUV deconvolution capabilities have been exploited to overcome coelution and finally separate individual signals.

Controlled release of glibenclamide from monolithic silica subdermal implants produced by the sol-gel process and its use for hyperglycaemia treatment in a murine model.

Glibenclamide is an anti-hyperglycaemic drug that is commonly used for the treatment of type 2 diabetes mellitus and has promising new medical indications. However, this drug is associated with high rates of serious hypoglycaemic episodes as a result of its pharmacological activity. Administering the drug through controlled release delivery systems could reduce the incidence of these episodes. In this study, glibenclamide silica monolithic xerogel implants for subdermal application (GMSIx) were developed using the sol-gel technique for matrix synthesis with TEOS with different drying conditions (environmental, 60, 90, and 120 °C, which were named as GMSIE, GMSI60, GMSI90, and GMSI120, respectively). The inclusion of the drug in monoliths was monitored by DSC, FTIR, and PXRD. The effect of drying conditions on the morphology, moisture content, hardness, dosage uniformity, surface characteristics, and drug release mechanism of glibenclamide from the matrices was systematically investigated. Oral Glucose Tolerance Tests were performed with mice to evaluate the efficacy of the GMSI in maintaining blood glucose levels. Glibenclamide was completely included in a non-crystalline solid form in the matrixes. The moisture content, hardness, dosage uniformity, and surface characteristics depend on the drying conditions. The monolithic matrices showed a mesoporous surface with high surface area, and a narrower pore size distribution occurred for GMSI60. GMSIE and GMSI60 showed non-Fickian anomalous Korsmeyer-Peppas glibenclamide release kinetics. GMSI90 and GMSI120 showed controlled release of the drug through dissolution. When GMSI60 was administered to mice, glucose blood levels were effectively maintained despite a high oral glucose load in the animals, showing a sustained effect of the drug released from this new sol-gel drug delivery system.

Molecular and Physiological Study of Candida albicans by Quantitative Proteome Analysis.

Candida albicans is one of the major pathogens that cause the serious infectious condition known as candidiasis. C. albicans was investigated by proteome analysis to systematically examine its virulence factors and to promote the development of novel pharmaceuticals against candidiasis. Here, we review quantitative time-course proteomics data related to C. albicans adaptation to fetal bovine serum, which were obtained using a nano-liquid chromatography/tandem mass spectrometry system equipped with a long monolithic silica capillary column. It was revealed that C. albicans induced proteins involved in iron acquisition, detoxification of oxidative species, energy production, and pleiotropic stress tolerance. Native interactions of C. albicans with macrophages were also investigated with the same proteome-analysis system. Simultaneous analysis of C. albicans and macrophages without isolating individual living cells revealed an attractive strategy for studying the survival of C. albicans. Although those data were obtained by performing proteome analyses, the molecular physiology of C. albicans is discussed and trials related to pharmaceutical applications are also examined.