Influence of peanut hairy root cultivars on prenylated stilbenoid production and the response mechanism for combining the elicitors of chitosan, methyl jasmonate, and cyclodextrin.

MAIN CONCLUSION: Peanut cultivars are known to produce stilbene compounds. Transcriptional control plays a key role in the early stages of the stress response mechanism, involving both PR-proteins and stilbene compounds. In this study, the production of stilbenoid compounds, especially prenylated, was investigated in two cultivars of peanut hairy root lines, designated as K2-K599 and T9-K599 elicited with a combination of chitosan (CHT), methyl jasmonate (MeJA), and cyclodextrin (CD): CHT + MeJA + CD. The antioxidant activities and stilbenoid content of both K2-K599 and T9-K599 hairy root lines increased significantly during the elicitation period. The T9-K599 hairy root line expressed higher ABTS and FRAP antioxidant activities than the K2-K599 line while the latter exhibited greater total phenolic content than the former at all-time points. Additionally, the K2-K599 line exhibited more stilbene compounds, including trans-resveratrol, trans-arachidin-1, and trans-arachidin-3 than the T9-K599 line, which showed statistically significant differences at all-time points. Gene expression of the enzyme involved in the stilbene biosynthesis pathway (PAL, RS, RS3) was observed, responding early to elicitor treatment and the metabolic production of a high level of stilbenoid compounds at a later stage. The antioxidant enzyme (CuZn-SOD, APX, GPX) and pathogenesis-related protein (PR; PR4A, PR5, PR10, chitinase) genes were strongly expressed after elicitor treatment at 24 h and decreased with an increasing elicitation time. Investigation of the response mechanism illustrates that the elicitor treatment can affect various plant responses, including plant cell wall structure and integrity, antioxidant system, PR-proteins, and secondary plant metabolites at different time points after facing external environmental stimuli.

Induction of the Prenylated Stilbenoids Arachidin-1 and Arachidin-3 and Their Semi-Preparative Separation and Purification from Hairy Root Cultures of Peanut (Arachis hypogaea L.).

Prenylated stilbenoids such as arachidin-1 and arachidin-3 are stilbene derivatives that exhibit multiple pharmacological activities. We report an elicitation strategy using different combinations of cyclodextrin, hydrogen peroxide, methyl jasmonate and magnesium chloride to increase arachidin-1 and arachidin-3 production in peanut hairy root cultures. The treatment of hairy root cultures with cyclodextrin with hydrogen peroxide selectively enhanced arachidin-1 yield (132.6 ± 20.4 mg/L), which was 1.8-fold higher than arachidin-3. Similarly, cyclodextrin combined with methyl jasmonate selectively enhanced arachidin-3 yield (178.2 ± 6.8 mg/L), which was 5.5-fold higher than arachidin-1. Re-elicitation of the hairy root cultures further increased the levels of arachidin-1 and arachidin-3 by 24% and 42%, respectively. The ethyl acetate extract of the culture medium was consecutively fractionated by normal- and reversed-phase column chromatography, followed by semi-preparative HPLC purification on a C18 column to yield arachidin-1 with a recovery rate of 32% and arachidin-3 with a recovery rate of 39%, both at higher than 95% purity. This study provided a sustainable strategy to produce high-purity arachidin-1 and arachidin-3 using hairy root cultures of peanuts combined with column chromatography and semi-preparative HPLC.

Amorphous Drug Solubility and Maximum Free Drug Concentrations in Cyclodextrin Solutions: A Quantitative Study Using NMR Diffusometry.

Cyclodextrin (CD) has been widely used as a solubilizing agent for poorly water-soluble drugs. In the present study, the effect of CD on the amorphous drug solubility and the maximum thermodynamic activity of the drug in the aqueous phase when the drug concentration exceeded the liquid-liquid phase separation (LLPS) concentration was investigated using three chemically diverse CDs, ß-cyclodextrin (ß-CD), dimethyl-ß-CD (DM-ß-CD), and hydroxypropyl-ß-CD (HP-ß-CD). The amorphous solubility of ibuprofen (IBP) increased substantially linearly with the increase in the CD concentration due to IBP/CD complex formation. Surprisingly, although the crystalline solubility of IBP in the ß-CD solution reached a plateau at ß-CD concentrations above 3 mM (BS-type solubility diagram) because of the limited crystalline solubility of the IBP/ß-CD complex, the amorphous solubility of IBP increased linearly even when the ß-CD concentration was higher than 3 mM. The amorphous solubility of IBP in CD solutions was influenced primarily by the phase separation of the IBP-supersaturated solution to the aqueous phase and the other phase mainly composed of IBP, namely, the IBP-rich phase, via LLPS. NMR spectroscopy revealed that DM-ß-CD was distributed into the IBP-rich phase when the IBP concentration exceeded its amorphous solubility, while ß-CD and HP-ß-CD showed minimal mixing with the IBP-rich phase. NMR diffusometry showed that the maximum free IBP concentration was reduced in the DM-ß-CD solution compared to that in the buffer. The mixing of DM-ß-CD with the IBP-rich phase reduced the chemical potential of IBP in the IBP-rich phase, which in turn reduced the maximum thermodynamic activity of IBP in the aqueous phase. In contrast, the maximum free IBP concentration was unchanged when ß-CD or HP-ß-CD was present. The hydrophobic nature of the DM-ß-CD substituent may contribute to its partitioning into the IBP-rich phase. The present study highlights the impact of CD on the maximum thermodynamic activity of drugs as well as the apparent amorphous solubility of the drug. This aspect should be considered for improving the effective absorption of poorly water-soluble drugs.

Direct distinction of ibuprofen and flurbiprofen enantiomers by ion mobility mass spectrometry of their ternary complexes with metal cations and cyclodextrins in the gas phase.

Enantiomeric drugs are widely used and play important roles in pharmaceuticals. Ion mobility spectrometry coupled with mass spectrometry technology provides a unique method for distinguishing the enantiomeric drugs, enantiomeric identification, and quantitation in the gas phase. In this study, enantiomeric molecules of ibuprofen and flurbiprofen were clearly recognized by forming host-guest complex ions using trapped ion mobility time-of-flight mass spectrometry. Ternary complex ions can be produced easily by electrospray ionization of the mixed solutions of ibuprofen, cyclodextrins, and CaCl2 , LiCl, or NaCl, as well as flurbiprofen, cyclodextrins, and CaCl2 . The relative contents of different chiral ibuprofens in a mixed solution were also quantitatively measured. This new method is a simple, effective, and a convenient enantioselective analysis method.

Hybrid MoS2/g-C3N4-assisted LDI mass spectrometry for rapid detection of small molecules and polyethylene glycols and direct determination of uric acid in complicated biological samples.

A novel matrix-assisted laser desorption/ionization time-of-flight mass spectrometric method (MALDI-TOF MS) for determination of highly sensitive small molecular compounds was developed based on molybdenum disulfide nanosheets hybridized with ultrathin graphitic carbon nitride (MoS2/g-C3N4) as the matrix. With this approach, the synergistic effects of MoS2 and g-C3N4 enhance the UV absorption of MoS2/g-C3N4, increase both desorption and ionization efficiency in LDI MS, and induce higher signal-to-noise ratio of analytes when compared with the bare MoS2 and g-C3N4 matrix in the determination of amino acids, antibiotics, neutral oligosaccharides, uric acid, and polyethylene glycols (PEGs). The detection limits of these small molecular compounds are in the ranges 0.1 to 10 µg mL-1, 1*10-3 to 1.0 µg mL-1, 1.0 to 10 µg mL-1, and 2*10-4 µg mL-1, respectively, and the polydispersity index of these PEGs is less than 1.02. Moreover, high salt tolera`nce and homogeneous deposition on the spot results in good reproducibility. The relative standard deviations (RSDs) of shot-to-shot and spot-to-spot (n = 15) of these compounds are less than 10.1% and 12.5%, respectively. With MoS2/g-C3N4, the uric acid in complicated biological samples can be directly determined in combination with LDI-TOF MS. We synthesized MoS2/g-C3N4 nanohybrid as an efficient matrix for MALDI-TOF MS analysis of small molecules as well as quantitative detection of uric acid in human urine.

Application of Molecular Dynamics Simulations in the Analysis of Cyclodextrin Complexes.

Cyclodextrins (CDs) are highly respected for their ability to form inclusion complexes via host-guest noncovalent interactions and, thus, ensofance other molecular properties. Various molecular modeling methods have found their applications in the analysis of those complexes. However, as showed in this review, molecular dynamics (MD) simulations could provide the information unobtainable by any other means. It is therefore not surprising that published works on MD simulations used in this field have rapidly increased since the early 2010s. This review provides an overview of the successful applications of MD simulations in the studies on CD complexes. Information that is crucial for MD simulations, such as application of force fields, the length of the simulation, or solvent treatment method, are thoroughly discussed. Therefore, this work can serve as a guide to properly set up such calculations and analyze their results.

Development of Spray-Dried Cyclodextrin-Based Pediatric Anti-HIV Formulations.

The human immunodeficiency virus (HIV) impacts up to 37 million people globally, of which 1.8 million are children. To date, there is no cure for HIV, although treatment options such as antiretroviral therapy (ART) are available. ART, which involves a patient taking a combination of antiretrovirals, is being used to treat HIV clinically. Despite the effectiveness of ART, there is currently no palatable pediatric formulation to treat HIV in children, which has hindered patient compliance and overall treatment efficacy. In addition, anti-HIV therapeutics are often poorly water-soluble, and hence have poor bioavailability. In the present study, we developed a pediatric-friendly formulation for anti-HIV therapeutics with improved dissolution characteristics of the therapeutic agents. Lopinavir (LPV) and ritonavir (RTV), available as FDA-approved fixed-dose combination products, were chosen as model ART drugs, and the formulation and processing parameters of spray-dried cyclodextrin (CD)-based LPV and RTV complexes were studied. Results showed that the spray-dried complexes exhibited enhanced dissolution profiles in comparison to pure drugs, particularly spray-dried ß-CD complexes, which showed the most favorable dissolution profiles. This current formulation with enhanced dissolution and taste-masking ability through the use of cyclodextrin has the potential to address the unmet need for the development of suitable pediatric formulations.

Active-electrode biosensor of SnO2 nanowire for cyclodextrin detection from microbial enzyme.

Cyclodextrin (CD) is a conical compound used in food and pharmaceutical industry to complexation of hydrophobic substances. It is a product of microbial enzymes which converts starch into CD during their activity. We aim to detect CD using active-electrode biosensor of SnO2. They were grown on active electrode by the VLS method. The CD consists of several glucose units which have hydroxyl groups which tend to bind to interface states present in nanowires changing their conductivity. Experimental results of electrical conductivity at different CD concentrations are presented. A model that describes the influence of adsorbed glucose on nanowires and its electrical properties is also presented. Some general observations are performed on the applicability of the CD adsorption method by the nanowire-based biosensor.

Mass Spectrometry as a Complementary Approach for Noncovalently Bound Complexes Based on Cyclodextrins.

An important and well-designed solution to overcome some of the problems associated with new drugs is provided by the molecular encapsulation of the drugs in the cyclodextrins (CDs) cavity, yielding corresponding inclusion complexes (ICs). These types of non-covalent complexes are of current interest to the pharmaceutical industry, as they improve the solubility, stability and bioavailability of the guest molecules. This review highlights several methods for cyclodextrin ICs preparation and characterization, focusing mostly on the mass spectrometry (MS) studies that have been used for the detection of noncovalent interactions of CDs inclusion complexes and binding selectivity of guest molecules with CDs. Furthermore, the MS investigations of several ICs of the CD with antifungal, antioxidants or fluorescent dyes are presented in greater details, pointing out the difficulties overcome in the analysis of this type of compounds.

Chiral Selectors in Capillary Electrophoresis: Trends During 2017⁻2018.

Chiral separation is an important process in the chemical and pharmaceutical industries. From the analytical chemistry perspective, chiral separation is required for assessing the fit-for-purpose and the safety of chemical products. Capillary electrophoresis, in the electrokinetic chromatography mode is an established analytical technique for chiral separations. A water-soluble chiral selector is typically used. This review therefore examines the use of various chiral selectors in electrokinetic chromatography during 2017⁻2018. The chiral selectors were both low and high (macromolecules) molecular mass molecules as well as molecular aggregates (supramolecules). There were 58 papers found by search in Scopus, indicating continuous and active activity in this research area. The macromolecules were sugar-, amino acid-, and nucleic acid-based polymers. The supramolecules were bile salt micelles. The low molecular mass selectors were mainly ionic liquids and complexes with a central ion. A majority of the papers were on the use or preparation of sugar-based macromolecules, e.g., native or derivatised cyclodextrins. Studies to explain chiral recognition of macromolecular and supramolecular chiral selectors were mainly done by molecular modelling and nuclear magnetic resonance spectroscopy. Demonstrations were predominantly on drug analysis for the separation of racemates.

Characterization of Inclusion Complex of Coenzyme Q10 with the New Carrier CD-MOF-1 Prepared by Solvent Evaporation.

The aim of the current study was to evaluate the physicochemical properties of a solid dispersion of coenzyme Q10 (CoQ10)/cyclodextrin metal organic frameworks-1 (CD-MOF-1). As a result of the powder X-ray diffraction (PXRD), it was confirmed that the CD-MOF-1 was changed from the α form to the ß form by evaporation (EVP). A diffraction peak due to melting of CoQ10 disappeared the EVP (CoQ10/CD-MOF-1 = 1/2). The structure of this complex is presumed to be similar to the ß form of CD-MOF-1. As a result of the differential scanning calorimetry (DSC), the endothermic peak due to the melting of CoQ10 disappeared the EVP (CoQ10/CD-MOF-1 = 1/2). As a result of the near-infrared (NIR) absorption spectroscopy, findings suggested the hydrogen bond in formation between the CH group in the isoprene side chains of CoQ10 and the OH group of CD-MOF-1. Therefore, the formation of crystal solid dispersion in CoQ10/CD-MOF-1 was suggested. As a result of the dissolution test in distilled water, the EVP (CoQ10/CD-MOF-1 = 1/2) had better dissolution in comparison to CoQ10 alone. Furthermore, also in fasted state simulated intestinal fluid (FaSSIF) in vivo, the EVP (CoQ10/CD-MOF-1 = 1/2) had better dissolution in the human body than CoQ10 alone. From the results of 2D-nuclear overhauser effect spectroscopy (NOESY) NMR spectroscopy, CD-MOF-1 could not include the benzoquinone ring of CoQ10. It was confirmed that the isoprene side chain was included. Therefore, it was suggested that CD-MOF-1 useful as a novel drug carrier for CoQ10.

Chiral selectors in CE: Recent development and applications (mid-2014 to mid-2016).

This report, which is a sequence of a series of reviews, records the most important chiral selectors (CSs) applied in CE. It highlights the CSs that were used during the period 2014 to mid-2016. In this review, method developments, validations, and pharmaceutical along with biomedical applications are presented. The different CSs include CDs, antibiotics, cyclofructants, linear and branched oligo- and polysaccharides, and polymeric surfactants. In addition, the advantages of these CSs, along with their chiral recognition mechanisms, and their performance, are discussed.

New agar microspheres for the separation and purification of natural products.

A new type of agar chromatography media has been prepared with a yield over 80% using a water-in-oil emulsion technique. These microspheres have regular spherical shapes and particle diameters in the range 40-165 µm (average ∼90 µm). Cross-linking of the resulting agar microspheres with epichlorohydrin and 1,4-butanediol diglycidyl ether enhanced their mechanical and thermal stability. The alkaline conditions used during the cross-linking reaction also decreased the content of ionized sulfate groups of the polysaccharide, thus reducing the nonspecific adsorption of positively charged molecules. The cross-linked agar microspheres were functionalized with (i) branched poly(ethyleneimine) to obtain a stationary phase useful for the separation of proteins in an anion-exchange mode and (ii) with poly-ß-cyclodextrin enabling direct isolation and purification of puerarin from a crude extract of Radix puerariae. Using a 23.5 mL column loaded with 20 mg extract (0.85 mg/mL gel), puerarin with a purity of 96% was recovered with a yield of 86%.

Effect of different levels of glycerol and cholesterol-loaded cyclodextrin on cryosurvival of ram spermatozoa.

This study was conducted to determine the optimum level of glycerol and cholesterol-loaded cyclodextrin (CLC) in a Tris-based diluent for cryopreservation of ram spermatozoa. Ram semen was treated with 0, 1.5, 3 or 4.5 mg CLC/120 × 10(6)  cells in Tris-based diluents containing 3, 5 or 7% glycerol in a factorial arrangement 3 × 4 and frozen in liquid nitrogen vapour. Sperm motility, viability (eosin-nigrosin staining) and functional membrane integrity (hypo-osmotic swelling test) were assessed immediately after thawing (0 h) and subsequently after 3 and 6 h at 37°C. There was an interaction between CLC and glycerol on the functional membrane integrity (p < 0.05). In the presence of 3% glycerol, the highest functional membrane integrity (32.2%) was found in the spermatozoa treated with 1.5 mg CLC/120 × 10(6)  sperm. Post-thaw sperm motility was highest in 1.5 mg CLC immediately after thawing (40.5%) and after 3-h (30.6%) incubation at 37°C (p < 0.05). Viability of spermatozoa was higher in all CLC treatments than in the untreated samples, and it was highest (33.9%) in the spermatozoa treated with 1.5 mg CLC (p < 0.05). These data indicate that the addition of cholesterol to sperm membranes by 1.5 mg CLC/120 × 10(6)  cells may allow the use of a lower concentration of glycerol (3%), which is sufficient to mitigate the detrimental effects of freezing and thawing.

Spectrofluorimetric study of estrogen-cyclodextrin inclusion complexes in aqueous systems.

For the first time, the spectrofluorimetric properties of estrone (E1), 17ß-estradiol (E2), estriol (E3), and 17α-ethinylestradiol (EE2) are studied in aqueous solutions after the addition of native and derivative cyclodextrins. In contrast to previous reports, the behavior of the systems is analysed in the absence of organic solvents able to modify the guest-host interaction. The significant differences between the obtained association constants with those reported in solvent mixtures are shown and discussed. In order to evaluate the influence of both the estrogen structure and the presence of cyclodextrin substituents on the inclusion phenomenon, fluorescent and acid-base behaviors of the systems are compared and discussed. The interaction of estrogens with micellar media formed by selected surfactants is also studied. It is demonstrated that estrogen-cyclodextrin complexes in aqueous solution are useful for improving fluorimetric detection limits and, since cyclodextrins are non-toxic and mitigate most of the solubility problems which require the use of organic solvents, the studied complexes are excellent candidates for extraction, separation, pre-concentration and removal processes maintaining the principles of the green analytical chemistry.

Interest of designed cyclodextrin-tools in gene delivery.

Cyclodextrins (CyDs) currently displays even today the image of a natural macrocyclic compound largely dominant in the formation of inclusion complexes with small hydrophobic molecules. During the past 10years, advances in this field allowed to achieve more and more sophisticated CyDs derivatives opening a simple access in scale-up quantities to original and better CyD-based gene delivery systems. In addition, possibility to combine covalent and supramolecular approaches offers new venues for the design of tailor-made CyD-based nanovehicles to improve their transfection ability and gene transfer in cells. In this account, we describe our recent progress in the construction of a novel CyD-based G0 (generation number) core dendrimer, scalable to CyD oligomers by a strategy using protonable guanidine tethers and whose concept can be generalized for the assembly of CyD pre-coated dendrimers. The synthetic strategy based on an original Staudinger-Aza-Wittig tandem coupling reaction. We present an outline of the different analytical strategies to characterize CyD-ODN (cyclodextrin-oligodeoxynucleotide) complexes. Among them, Capillary electrophoresis (CE) was used to perfectly characterize our CyD-siRNA and CyD-DNA complexes and shown to be a very attractive method with advantages of low sample consumption, rapid analysis speed, and high efficiency that make this technology a major tool for association constant measurement. Finally, we present the different biological methods that can be used, in vitro, to study gene delivery, and more precisely ones we have performed to evaluate the capability of our original model bis-guanidinium-tetrakis-ß-cyclodextrin dendrimeric tetrapod, to deliver efficiently DNA or siRNA in eukaryotic cells.

Analysis of a polydisperse polyrotaxane based on poly(ethylene oxide) and α-cyclodextrins using nanoelectrospray and LTQ-Orbitrap.

Polyrotaxanes (PR) are among the most studied interlocked molecules in the field of supramolecular chemistry. Cyclodextrin based polyrotaxanes (CD based PRs) are well-known to be difficult to analyze by mass spectrometry (MS). Nanoelectrospray (nanoESI) employed during mass spectrometry (MS) and tandem mass spectrometry (MS/MS) experiments turns out to be particularly useful to analyze these noncovalent assemblies. While ESI/nanoESI based spectra usually contain multicharged species which greatly complicate the interpretation, particularly for such complex mixtures analysis, the hyphenation with a high resolution analyzer such as Orbitrap could overcome this limitation. This Article reports efforts to achieve a detailed structural deciphering by nanoESI-MS and nanoESI-MS/MS of CD based PRs constituted of αCDs, unmodified or surrounded by 1 or 2 sulfation(s), which were threaded along polydisperse poly(ethylene oxide) α,ω-dipyrenyl chains. The described method is more sensitive and less sample consuming than a typical NMR experiment and in good agreement with size-exclusion chromatography (SEC) results. Moreover, as compared to MALDI-TOF MS analysis, all populations were presumably elucidated without discrimination effect. Therefore, this MS development allowed us to estimate the PR sample content with 16 to 35 ethylene oxide units, 1 to 5 αCDs threaded, and 0 to 10 sulfo groups grafted on the overall CDs. Finally, the method afforded the possibility to unambiguously attribute supramolecular architectures from 2276.0278 to 7767.8342 g·mol(-1) corresponding to poly[2]- to poly[6]rotaxanes.

Preparation of a novel cyclodextrin derivative of benzimido-ß-cyclodextrin and its enantioseparation performance in HPLC.

A novel cyclodextrin (CD) derivative, mono-6-deoxy-benzimide-ß-CD (MB-ß-CD), in which a rigid substituent was linked to the narrow edge of the CD with a flexible H(2)C-N group, was successfully synthesized through the condensation of mono-6-deoxy-6-amino-ß-cyclodextrin and benzaldehyde. To evaluate its enantioseparation abilities and investigate the role of the CD substituents and linkage in chiral recognition, MB-ß-CD and mono-6-deoxyphenylimine-ß-CD (MP-ß-CD) with a rigid linkage were compared in the separation of 36 chiral compounds in a methanol/water mobile phase. The separation results showed that most of the analytes with rigid structures afforded better enantioresolutions on the MP-ß-CD (with a rigid linkage) chiral stationary phase (CSP), while better enantioseparations for analytes with flexible structures and big steric hindrance were obtained on the MB-ß-CD (with a flexible linkage) CSP. The former exhibited a specificity for the analyte structures, while the latter was more adaptable. Molecular dynamics simulations were performed to further understand the discrimination process and the function of the CD side arm.

Negatively charged cyclodextrins: Synthesis and applications in chiral analysis-A review.

The negatively charged cyclodextrins (CDs) play an important role in chiral analysis due to the additional electrostatic effect beyond the host-guest inclusion, especially in enantioanalysis of positively charged and electrically neutral analytes. This review presents recent advances in application of anionic CDs for enantioanalysis during the past five years. Firstly, the synthesis approaches of random substitution and single isomers of anionic CDs are briefly discussed. The main part focuses on the chiral analysis using anionic CDs in various analytical techniques, including capillary electrophoresis, high-performance liquid chromatography, capillary electrochromatography, counter current chromatography, nuclear magnetic resonance, etc. Particular attention is given to the capillary electrophoresis application since charged CDs could be used as a carrier of enantiomers by virtue of their self-mobility and offer an easy adjustment of the enantiomer migration order. Finally, future opportunities are also discussed in the conclusion of this review.

Multifunctional ZrO(2) nanoparticles and ZrO(2)-SiO (2) nanorods for improved MALDI-MS analysis of cyclodextrins, peptides, and phosphoproteins.

Multifunctional ZrO(2) nanoparticles (NPs) and ZrO(2)-SiO(2) nanorods (NRs) have been successfully applied as the matrices for cyclodextrins and as affinity probes for enrichment of peptides (leucine-enkephalin, methionine-enkephalin and thiopeptide), phosphopeptides (from tryptic digestion products of beta-casein) and phosphoproteins from complex samples (urine and milk) in atmospheric pressure matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and MALDI time-of-flight (TOF) MS. The results show that the ZrO(2) NPs and ZrO(2)-SiO(2) NRs can interact with target molecules (cyclodextrins, peptides, and proteins), and the signal intensities of the analytes were significantly improved in MALDI-MS. The maximum signal intensities of the peptides were obtained at pH 4.5 using ZrO(2) NPs and ZrO(2)-SiO(2) NRs as affinity probes. The limits of detection of the peptides were found to be 75-105 fmol for atmospheric pressure MALDI-MS and those of the cyclodextrins and beta-casein were found to be 7.5-20 and 115-125 fmol, respectively, for MALDI-TOF-MS. In addition, these nanomaterials can be applied as the matrices for the analysis of cyclodextrins in urine samples by MALDI-TOF-MS. ZrO(2) NPs and ZrO(2)-SiO(2) NRs efficiently served as electrostatic probes for peptide mixtures and milk proteins because 2-11 times signal enhancement can be achieved compared with use of conventional organic matrices. Moreover, we have successfully demonstrated that the ZrO(2) NPs can be effectively applied for enrichment of phosphopeptides from tryptic digestion of beta-casein. Comparing ZrO(2) NPs with ZrO(2)-SiO(2) NRs, we found that ZrO(2) NPs exhibited better affinity towards phosphopeptides than ZrO(2)-SiO(2) NRs. Furthermore, the ZrO(2) and ZrO(2)-SiO(2) nanomaterials could be used to concentrate trace amounts of peptides/proteins from aqueous solutions without tedious washing procedures. This approach is a simple, straightforward, separation-and washing-free approach for MALDI-MS analysis of cyclodextrins, peptides, proteins, and tryptic digestion products of phosphoproteins.