Characterization by LC-MS/MS of oxidized products identified in synthetic peptide somatostatin and cetrorelix submitted to forced oxidative stress by hydrogen peroxide: Two case studies.

In a broader scenario, the forced degradation studies provided by the ICH guidelines for Q1A, Q1B, and Q2B degradation studies allow to know the CQA of the molecule used as a drug product, to determine the appropriate analytical methods, excipients, and storage conditions ensuring the quality of the drug, its efficacy, and patient safety. In this study, we focused our attention on understanding how oxidative stress is performed by H2 O2 -impacted small synthetic peptides that do not contain residues susceptible to oxidation such as methionine. Among the amino acids susceptible to oxidation, methionine is the most reactive and depending on the structure of the protein where it is exposed, it tends to oxidize by converting into methionine sulfone or methionine sulfoxide by oxidation of its sulfur atom. Scouting experiments obtained by forced oxidative stress conditions are presented on two small synthetic peptides that do not contain any methionine residues spiked with different amounts of H2 O2 , and they are analyzed by LC-MS/MS. Less frequent oxidation products than those commonly observed on proteins/peptides-containing methionine have been characterized on both peptides. The study demonstrated that somatostatin, by means of one residue of tryptophan on the molecule, can generate traces of several oxidized products detected by UPLC-MS. Furthermore, even at a negligible level, oxidation on tyrosine and proline in cetrorelix that does not contain methionine nor tryptophan has been detected by UHPLC-MS/MS. Identification and quantification of oxidized species were achieved by high-resolution MS and MS/MS experiments. Thus, FDSs undoubtedly aid the evaluation of the CQAs as an important component of the characterization package as recommended by HAs and ICH, facilitating the understanding of unforeseen features of the studied molecule used as drugs.

Capillary methacrylate-based monoliths by grafting from/to γ-ray polymerization on a tentacle-type reactive surface for the liquid chromatographic separations of small molecules and intact proteins.

Capillary methacrylate-based monoliths were prepared for the high performance liquid chromatography (HPLC) separation of both small molecules and large biomolecules. An efficient grafting from/to synthetic approach was adopted introducing a network of activated sites in the inner wall surface using the new silanization agent (N-trimethoxysilylpropyl)-polyethylenimine. Copolymerization of lauryl methacrylate monomer and 1,6-hexanediol dimethacrylate cross-linker in the presence of porogenic solvents was obtained under continuous γ-ray exposure with high conversion yield. The morphology and porous structure of the resulting monoliths have been investigated by Scanning Electron Microscopy (SEM) and 1H NMR cryoporosimetry. By chromatographic investigation, the new capillary columns attested high kinetic performance (with efficiency larger than 100,000 theoretical plate/m for small molecules at optimum mobile phase linear velocity of about 0.5mm/s) and also excellent mechanical stability and repeatability. The new methacrylate-based monolithic capillary columns have been successfully employed for efficient reversed-phase separation of intact proteins and peptides.

Separation of intact proteins on γ-ray-induced polymethacrylate monolithic columns: A highly permeable stationary phase with high peak capacity for capillary high-performance liquid chromatography with high-resolution mass spectrometry.

Polymethacrylate-based monolithic capillary columns, prepared by γ-radiation-induced polymerization, were used to optimize the experimental conditions (nature of the organic modifiers, the content of trifluoroacetic acid and the column temperature) in the separation of nine standard proteins with different hydrophobicities and a wide range of molecular weights. Because of the excellent permeability of the monolithic columns, an ion-pair reversed-phase capillary liquid chromatography with high-resolution mass spectrometry method has been developed by coupling the column directly to the mass spectrometer without a flow-split and using a standard electrospray interface. Additionally, the high working flow and concomitant high efficiency of these columns allowed us to employ a longer column (up to 50 cm) and achieve a peak capacity value superior to 1000. This work is motivated by the need to develop new materials for high-resolution chromatographic separation that combine chemical stability at elevated temperatures (up to 75°C) and a broad pH range, with a high peak capacity value. The advantage of the γ-ray-induced monolithic column lies in the batch-to-batch reproducibility and long-term high-temperature stability. Their proven high loading capacity, recovery, good selectivity and high permeability, moreover, compared well with that of a commercially available poly(styrene-divinylbenzene) monolithic column, which confirms that such monolithic supports might facilitate analysis in proteomics.

Revealing the fine details of functionalized silica surfaces by solid-state NMR and adsorption isotherm measurements: the case of fluorinated stationary phases for liquid chromatography.

The structural and chromatographic characterization of two novel fluorinated mesoporous materials prepared by covalent reaction of 3-(pentafluorophenyl)propyldimethylchlorosilane and perfluorohexylethyltrichlorosilane with 2.5 µm fully porous silica particles is reported. The adsorbents were characterized by solid state (29)Si, (13)C, and (19)F NMR spectroscopy, low-temperature nitrogen adsorption, elemental analysis (C and F), and various chromatographic measurements, including the determination of adsorption isotherms. The structure and abundance of the different organic surface species, as well as the different silanol types, were determined. In particular, the degree of so-called horizontal polymerization, that is, Si-O-Si bridging parallel to the silica surface due to the reaction, under "quasi-dry" conditions, of trifunctional silanizing agents with the silica surface was quantified. Significant agreement was found between the information provided by solid-state NMR, elemental analysis, and excess isotherms regarding the amount of surface residual silanol groups, on the one hand, and the degree of surface functionalization, on the other. Finally, the kinetic performance of the fluorinated materials as separation media for applications in near-ultrahigh-performance liquid chromatography was evaluated. At reduced velocities of about 5.5 (ca. 600 bar backpressure at room temperature) with 3 mm diameter columns and toluene as test compound, reduced plate heights on the order of 2 were obtained on columns of both adsorbents.

Analysis of bovine milk caseins on organic monolithic columns: an integrated capillary liquid chromatography-high resolution mass spectrometry approach for the study of time-dependent casein degradation.

Casein proteins constitute approximately 80% of the proteins present in bovine milk and account for many of its nutritional and technological properties. The analysis of the casein fraction in commercially available pasteurized milk and the study of its time-dependent degradation is of considerable interest in the agro-food industry. Here we present new analytical methods for the study of caseins in fresh and expired bovine milk, based on the use of lab-made capillary organic monolithic columns. An integrated capillary high performance liquid chromatography and high-resolution mass spectrometry (Cap-LC-HRMS) approach was developed, exploiting the excellent resolution, permeability and biocompatibility of organic monoliths, which is easily adaptable to the analysis of intact proteins. The resolution obtained on the lab-made Protein-Cap-RP-Lauryl-γ-Monolithic column (270 mm × 0.250 mm length × internal diameter, L × I.D.) in the analysis of commercial standard caseins (αS-CN, ß-CN and κ-CN) through Cap-HPLC-UV was compared to the one observe using two packed capillary C4 columns, the ACE C4 (3 µm, 150 mm × 0.300 mm, L × I.D.) and the Jupiter C4 column (5 µm, 150 mm × 0.300 mm, L × I.D.). Thanks to the higher resolution observed, the monolithic capillary column was chosen for the successive degradation studies of casein fractions extracted from bovine milk 1-4 weeks after expiry date. The comparison of the UV chromatographic profiles of skim, semi-skim and whole milk showed a major stability of whole milk towards time-dependent degradation of caseins, which was further sustained by high-resolution analysis on a 50-cm long monolithic column using a 120-min time gradient. Contemporarily, the exact monoisotopic and average molecular masses of intact αS-CN and ß-CN protein standards were obtained through high resolution mass spectrometry and used for casein identification in Cap-LC-HRMS analysis. Finally, the proteolytic degradation of ß-CN in skim milk and the contemporary formation of low-molecular-weight proteose-peptones (PP) with exact monoisotopic Mr between 9444.0989 Da and 14098.9861 Da was confirmed through the deconvolution of high resolution mass spectra and literature data.

Stereolability of dihydroartemisinin, an antimalarial drug: a comprehensive kinetic investigation. Part 2.

Artemisinin or qinghaosu has now largely given way to the more potent dihydroartemisinin (DHA, 1) and its derivatives in the treatment of drug-resistant malaria, in combination with other classical antimalarial drugs. DHA is obtained by NaBH(4) reduction of artemisinin and contains a stereochemically labile center at C-10, which provided two lactol hemiacetal interconverting epimers, namely 1α and 1ß. In the solid state, the drug consists exclusively of the ß-epimer; however, upon dissolution, the two epimers equilibrate, reaching different solvent-dependent ratios with different rates. Such equilibration also occurs in vivo, irrespective of the isomeric purity at which the drug would have been administered. The aim of this study was then to achieve an in-depth understanding of the kinetic features of the α/ß equilibration. To this purpose, free energy activation barriers (ΔG(‡)) of the interconversion were determined as a function of both general and specific acid and base catalysts, ionic strength, and temperature in different solvents by dynamic HPLC (DHPLC). In hydro-organic media, the dependence of ΔG(‡) on temperature led to the evaluation of the related enthalpic and entropic contributions. Theoretical calculations suggested that the rate-determining step of the interconversion is not the ring-opening of the cyclic hemiacetal but the previous reversible deprotonation of the individual epimers (base-catalyzed mechanism). The whole findings may contribute to shed some light on the mechanism of action and/or bioavailability of the drug at the molecular level.

Efficient organic monoliths prepared by γ-radiation induced polymerization in the evaluation of histone deacetylase inhibitors by capillary(nano)-high performance liquid chromatography and ion trap mass spectrometry.

New monolithic HPLC columns were prepared by γ-radiation-triggered polymerization of hexyl methacrylate and ethylene glycol dimethacrylate monomers in the presence of porogenic solvents. Polymerization was carried out directly within capillary (250-200 µm I.D.) and nano (100-75 µm I.D.) fused-silica tubes yielding highly efficient columns for cap(nano)-LC applications. The columns were applied in the complete separation of core (H2A, H2B, H3, and H4) and linker (H1) histones under gradient elution with UV and/or electrospray ionization (ESI) ion trap mass spectrometry (MS) detections. Large selectivity towards H1, H2A-1, H2A-2, H2B, H3-1, H3-2 and H4 histones and complete separation were obtained within 8 min time windows, using fast gradients and very high linear flow velocities, up to 11 mm/s for high throughput applications. The method developed was the basis of a simple and efficient protocol for the evaluation of post-translational modifications (PTMs) of histones from NCI-H460 human non-small-cell lung cancer (NSCLC) and HCT-116 human colorectal carcinoma cells. The study was extended to monitoring the level of histone acetylation after inhibition of Histone DeACetylase (HDAC) enzymes with suberoylanilide hydroxamic acid (SAHA), the first HDAC inhibitor approved by the FDA for cancer therapy. Attractive features of our cap(nano)-LC/MS approach are the short analysis time, the minute amount of sample required to complete the whole procedure and the stability of the polymethacrylate-based columns. A lab-made software package ClustMass was ad hoc developed and used to elaborate deconvoluted mass spectral data (aligning, averaging, clustering) and calculate the potency of HDAC inhibitors, expressed through a Relative half maximal Inhibitory Concentration parameter, namely R_IC(50) and an averaged acetylation degree.

Stereolability of dihydroartemisinin, an antimalarial drug: a comprehensive thermodynamic investigation. Part 1.

Artemisinin (Qinghaosu, 1) is a sesquiterpene lactone endoperoxide isolated from Artemisia annua L. that Chinese herbalists have traditionally used to treat malaria. Reduction of artemisinin by NaBH(4) produced dihydroartemisinin (DHA, 2) and yielded a new stereochemically labile center at C-10, which in turn provided two lactol hemiacetal interconverting epimers, namely, 2α and 2ß. With the aim of fully investigating the thermodynamics of interconversion, we gathered the relative abundance of the two epimers within a wide variety of solvents and rationalized the results by linear solvation energy relationships (LSER) analysis. Beside the difference in polarity, the better stabilization of 2α in polar solvents was found to be significantly related to its greater acidity with respect to 2ß, which was estimated by two independent theoretical approaches based on molecular modeling calculations and empirical data, and supported by (1)H NMR measurements. On the contrary, differential effects of cavitational energy have been highlighted as interactions strongly responsible for the small values of equilibrium constant measured for the ß â‡† α process in the less polar media. Determination of forward and backward epimerization rate constants in seven media, clearly differing in both permittivity and capacity to be H-bond donors, indicated that, in the spontaneous process, the transition state of the rate-limiting step develops a significant degree of anionic character, as typically happens in the base-catalyzed breakdown of hemiacetals.

Analysis of aluminium content and iron homeostasis in nipple aspirate fluids from healthy women and breast cancer-affected patients.

Aluminium is not a physiological component of the breast but has been measured recently in human breast tissues and breast cyst fluids at levels above those found in blood serum or milk. Since the presence of aluminium can lead to iron dyshomeostasis, levels of aluminium and iron-binding proteins (ferritin, transferrin) were measured in nipple aspirate fluid (NAF), a fluid present in the breast duct tree and mirroring the breast microenvironment. NAFs were collected noninvasively from healthy women (NoCancer; n = 16) and breast cancer-affected women (Cancer; n = 19), and compared with levels in serum (n = 15) and milk (n = 45) from healthy subjects. The mean level of aluminium, measured by ICP-mass spectrometry, was significantly higher in Cancer NAF (268.4 ± 28.1 µg l(-1) ; n = 19) than in NoCancer NAF (131.3 ± 9.6 µg l(-1) ; n = 16; P < 0.0001). The mean level of ferritin, measured through immunoassay, was also found to be higher in Cancer NAF (280.0 ± 32.3 µg l(-1) ) than in NoCancer NAF (55.5 ± 7.2 µg l(-1) ), and furthermore, a positive correlation was found between levels of aluminium and ferritin in the Cancer NAF (correlation coefficient R = 0.94, P < 0.001). These results may suggest a role for raised levels of aluminium and modulation of proteins that regulate iron homeostasis as biomarkers for identification of women at higher risk of developing breast cancer. The reasons for the high levels of aluminium in NAF remain unknown but possibilities include either exposure to aluminium-based antiperspirant salts in the adjacent underarm area and/or preferential accumulation of aluminium by breast tissues.

Iron-binding proteins and C-reactive protein in Nipple Aspirate Fluids: role of Iron-driven inflammation in breast cancer microenvironment?

Breast cancer, a worldwide disease with increasing incidence, develops from ductal/lobular epithelium. Nipple aspirate fluid (NAF), secreted from the breast ducts and lobules, can be analyzed to assess metabolic activity in breast microenvironment. Premalignant and malignant cell alterations may produce biochemical signals that deliver inflammatory proteins to the site. C-reactive protein (CRP), acute-phase protein considered a prognostic marker of inflammation, is frequently over-expressed in invasive breast carcinomas. Starting from the evidence that soluble and cell-bound iron binding protein Ferritin (FTN) and Transferrin (TRF) are crucially involved in breast inflammation and cancer, the aim of the present study is to analyze in NAF (a ductal fluid mirroring the breast microenvironment noninvasively collected from healthy and proven breast cancer affected women, n=38), the concentrations of CRP, FTN and TRF through high sensitive immunoassays. We analysed also serum (n=35) and milk samples (n=20) from healthy subjects. The mean level of CRP in Cancer NAF was significantly higher than in NoCancer NAF (P < 0.0001), especially in postmenopausal patients. Moreover, in Cancer NAF we detected higher levels of TRF and FTN respect to NoCancer NAF (P<0.001). A highly significant positive correlation between FTN and CRP content (Y= 2322x + 6.196, r(2) = 0.651, P<0.0001) was found. These data may support the involvement of inflammation and deregulation of iron homeostasis in breast cancer etio-pathogenesis. The significant accumulation of CRP in NAF in conjunction to the disruption of iron homeostasis may help to identify women at higher breast cancer risk.

Hybrid polyacrylamide chiral stationary phases for HPLC prepared by surface-initiated photopolymerization.

Two hybrid polyacrylamide chiral stationary phases (CSPs) for HPLC have been synthesized by a new surface-initiated photo-induced radical polymerization approach of enantiopure N,N'-diacryloyl derivatives of (1R,2R)-diaminocyclohexane (CSP1) and (1R,2R)-diphenylethylenediamine (CSP2). This system is based on the activation of mesoporous silica microparticles by chemically bonded trichloroacetyl groups and dimanganese decacarbonyl as catalyst. UV irradiation was performed using a lab-made quartz photochemical reactor, ad hoc designed for the photo-induced polymerization process on the surface of microparticles. The two phases were evaluated and compared as chromatographic supports for the enantioselective HPLC of model chiral compounds. Their physico-chemical properties and chromatographic performances were also evaluated in comparison with those exhibited by the homologue CSPs obtained by the grafting-from thermal-induced process (CSP3 and CSP4). The new photopolymerization approach yielded higher grafting density than the thermal-induced one, especially in the case of the less reactive monomer (the diacryloyl derivative of (1R,2R)-diphenylethylenediamine), good chromatographic efficiency and a broad application field under normal phase and polar organic mode conditions.

Detection of superoxide dismutase-1 in nipple aspirate fluids: a reactive oxygen species-regulating enzyme in the breast cancer microenvironment.

INTRODUCTION: Analysis of nipple aspirate fluid (NAF) allows the noninvasive identification of both cellular and biomolecular markers of human breast cancer, the most common female malignancy in developed countries. Cytosolic superoxide dismutase (SOD-1) represents a detoxifying enzyme able to regulate the balance between oncogenic and oncosuppressor reactive oxygen species. PATIENTS AND METHODS: We analyzed SOD-1 expression in 126 NAF samples collected from 67 women with and 59 without breast cancer, using enzyme-linked immunosorbent assay, immunoprecipitation, Western blotting, and immunocytochemistry. RESULTS: SOD-1 median values in plasma presented no difference between the no-cancer and cancer subgroups. No significant difference in the median level of SOD-1 between matched plasma and NAF from cancer patients was found, whereas SOD-1 median level in no-cancer NAF was significantly higher when compared with matched plasma. Finally, the SOD-1 median value in no-cancer NAF was significantly higher (about 2-fold) than in cancer NAF. CONCLUSION: NAF measurement of SOD-1 is a useful tool to identify intracellular redox status of the breast microenvironment, mirroring the oxidative metabolic pathways occurring in breast tissue, both in physiologic and cancer conditions and in improving the identification of women at increased breast cancer risk. SOD-1 activity in the breast microenvironment may represent a functional "switch" between the detrimental/oncogenic properties and the oncosuppressor/proapoptotic role of this antioxidant enzyme.

Transition from enantioselective high performance to ultra-high performance liquid chromatography: A case study of a brush-type chiral stationary phase based on sub-5-micron to sub-2-micron silica particles.

Three brush-type chiral stationary phases (CSPs) differing in the particle size of the starting silica particles have been prepared by covalent grafting of the pi-acidic bis-(3,5-dinitrobenzoyl)-derivative of trans-1,2-diaminocyclohexane (DACH-DNB). Starting silica particles of 4.3, 2.6 and 1.9micron were used to generate the final CSPs using an improved, highly reproducible synthetic methodology, that allowed to assemble and surface-graft the whole chiral selector in only two steps. The different CSPs have been packed in columns of various length and diameters, and fully characterized in terms of flow permeability, kinetic performances and enantioselectivity using a set of test solutes. Very high speed and high resolution applications together with stereodynamic HPLC examples are demonstrated on the columns with reduced particle diameters, on which separations of several enantiomeric pairs are routinely obtained with analysis times in the 15-40s range.

On-column epimerization of dihydroartemisinin: an effective analytical approach to overcome the shortcomings of the International Pharmacopoeia monograph.

We developed a cryo-HPLC/UV method for the simultaneous determination of artemisinin (1), alpha-dihydroartemisinin (2alpha), beta-dihydroartemisinin (2beta), and a ubiquitous thermal decomposition product of 2 (designated as diketoaldehyde, 3), starting from the International Pharmacopoeia monograph on dihydroartemisinin. The method takes for the first time the on-column epimerization process of 2 into consideration. Chromatographic separation was obtained under reversed-phase conditions on a Symmetry C18 column (3.5 microm particle size) with a mobile phase consisting of acetonitrile-water 60:40 (v/v), delivered at 0.60-1.00 ml/min flow-rates, with ultraviolet detection at low wavelength (lambda = 210 nm). Low temperatures (T = 0-10 degrees C) were selected on the grounds of a diastereoselective dynamic HPLC (DHPLC) study performed at different temperatures, aimed at identifying the best experimental conditions capable of minimizing the on-column interconversion process.