Online trapping and enrichment ultra performance liquid chromatography–tandem mass spectrometry method for sensitive measurement of “arginine-asymmetric dimethylarginine cycle” biomarkers in human exhaled breath condensate

Background

Exhaled breath condensate (EBC) is a biofluid collected non invasively that, enabling the measurement of several biomarkers, has proven useful in the study of airway inflammatory diseases, including asthma, COPD and cystic fibrosis. To the best of our knowledge, there is no previous report of any analytical method to detect ADMA in EBC.

Objectives

Aim of this work was to develop an online sample trapping and enrichment system, coupled with an UPLC–MS/MS method, for simultaneous quantification of seven metabolites related to “Arginine-ADMA cycle”, using the isotopic dilution.

Methods

Butylated EBC samples were trapped in an online cartridge, washed before and after each injection with cleanup solution to remove matrix components and switched inline into the high pressure analytical column. Multiple reaction monitoring in positive mode was used for analyte quantification by tandem mass spectrometry.

Results

Validation studies were performed in EBC to examine accuracy, precision and robustness of the method. For each compound, the calibration curves showed a coefficient of correlation (r²) greater than 0.992. Accuracy (%Bias) was <3% except for NMMA and H-Arg (<20%), intra- and inter-assay precision (expressed as CV%) were within ±20% and recovery ranged from 97.1 to 102.8% for all analytes.Inter-day variability analysis on 20 EBC of adult subjects did not demonstrate any significant variation of quantitative data for each metabolite. ADMA and SDMA mean concentrations (μmol L⁻¹), measured in EBC samples of asthmatic adolescents are significantly increased (p < 0.0001) than in normal controls (0.0040 ± 0.0021 vs. 0.0012 ± 0.0005 and 0.0020 ± 0.0015 vs. 0.0002 ± 0.0001, respectively), as well the ADMA/Tyr (0.34 ± 0.09 vs. 0.12 ± 0.02, p < 0.0001) and the SDMA/Tyr ratio (0.10 ± 0.04 vs. 0.015 ± 0.004, p < 0.0001).

Conclusions

The proposed method features simple specimen preparation, maintenance of an excellent peak shape of all metabolites and reduced matrix effects as well mass spectrometer noise. Moreover, the possibility to perform different cycles of enrichment, using large injection volumes, compensated for the low concentration of analytes contained in EBC, leading to a good analytical sensitivity. Preliminary data obtained from asthmatic and healthy adolescents, demonstrated that the analytical method applied to EBC seems suitable not only for research purposes, but also for clinical routinely analysis.     

Enantioseparation of Chiral Antimycotic Drugs by HPLC with Polysaccharide-Based Chiral Columns and Polar Organic Mobile Phases with Emphasis on Enantiomer Elution Order

The separation of enantiomers of 10 chiral antimycotic drugs was studied on polysaccharide-based chiral columns with polar organic mobile phases. The emphasis was placed on some interesting examples of enantiomer elution order reversal observed depending on the chemistry of the chiral selector, separation temperature, major component, as well as the minor additive in the mobile phase. In particular, it was found that the elution order of enantiomers of chiral drug terconazole was opposite on cellulose- and amylose-based columns with the same pendant group. The affinity pattern of enantiomers of another chiral drug bifonazole was opposite towards to two amylose-based chiral selectors with different pendant groups. The affinity pattern of terconazole enantiomers also changed on some columns when the alcohol-based mobile phase was replaced with acetonitrile. An interesting effect of the minor acidic (formic acid) additives to the mobile phase on the affinity pattern of terconazole enantiomers was observed on Cellulose-2 and Cellulose-4 columns. In addition, a reversal of elution order of bifonazole enantiomers was observed on Amylose-2 column by variation of a separation temperature.

     

Atheroprotective Effects of Glycyrrhiza glabra L.

Cardiovascular diseases associated with atherosclerosis are the major cause of death in developed countries. Early prevention and treatment of atherosclerosis are considered to be an important aspect of the therapy of cardiovascular disease. Preparations based on natural products affect the main pathogenetic steps of atherogenesis, and so represent a perspective for the long-term prevention of atherosclerosis development. Numerous experimental and clinical studies have demonstrated the multiple beneficial effects of licorice and its bioactive compounds—anti-inflammatory, anti-cytokine, antioxidant, anti-atherogenic, and anti-platelet action—which allow us to consider licorice as a promising atheroprotective agent. In this review, we summarized the current knowledge on the licorice anti-atherosclerotic mechanisms of action based on the results of experimental studies, including the results of the in vitro study demonstrating licorice effect on the ability of blood serum to reduce intracellular cholesterol accumulation in cultured macrophages, and presented the results of clinical studies confirming the ameliorating activity of licorice in regard to traditional cardiovascular risk factors as well as the direct anti-atherosclerotic effect of licorice.     

A Computational Quantum-Based Perspective on the Molecular Origins of Life’s Building Blocks

The search for the chemical origins of life represents a long-standing and continuously debated enigma. Despite its exceptional complexity, in the last decades the field has experienced a revival, also owing to the exponential growth of the computing power allowing for efficiently simulating the behavior of matter—including its quantum nature—under disparate conditions found, e.g., on the primordial Earth and on Earth-like planetary systems (i.e., exoplanets). In this minireview, we focus on some advanced computational methods capable of efficiently solving the Schrödinger equation at different levels of approximation (i.e., density functional theory)—such as ab initio molecular dynamics—and which are capable to realistically simulate the behavior of matter under the action of energy sources available in prebiotic contexts. In addition, recently developed metadynamics methods coupled with first-principles simulations are here reviewed and exploited to answer to old enigmas and to propose novel scenarios in the exponentially growing research field embedding the study of the chemical origins of life.     

HPLC separation of dihydropyridine derivatives enantiomers with emphasis on elution order using polysaccharide‐based chiral columns

The separation of enantiomers of five chiral dihydropyridine derivatives was studied on five different polysaccharide‐based chiral HPLC columns with various normal‐phase (NP), polar organic, and reversed‐phase eluents. Along with the successful separation of analyte enantiomers, the emphasis of this study was on enantiomer elution order (EEO) with various columns and mobile phase composition. The interesting phenomenon of reversal of EEO, recently reported in the case of amlodipine (AML) depending on the concentration of formic acid in acetonitrile, was also confirmed with NP eluents. Under RP conditions at relatively low water content, the EEO of AML could also be reverted by varying the concentration of formic acid in the mobile phase. However, at higher water content the same parameter did not affect the EEO, but only induced gradual decrease in resolution up to complete co‐elution of enantiomers. Additionally, in organic‐aqueous mobile phases retention factors decreased with increasing water content but only up to 20% (v/v), while above this concentration the expected typical RP behavior was observed. The presence of the commonly used additive diethylamine in the mobile phase seems important for observing a reversal in EEO with increasing concentration of formic acid. The reversal of the EEO was characteristic of AML only and was not observed for any of other dihydropyridines included in this study.     

Influence of nutrient medium composition on in vitro growth, polyphenolic content and antioxidant activity of Alchemilla mollis

Alchemilla mollis (Buser) Rothm. (Rosaceae) is a high-mountain medicinal plant growing in the Balkan Peninsula, with only one population in Bulgaria. Alchemilla plants (Lady's mantle) are commonly used in traditional medicine for treatment of many gynecological diseases. The commercial drugs "Herba Alchemillae" induce a rapid regeneration of skin epithelium and have styptic and anti-inflammatory actions. Because of the high content of phenolic compounds (tannins and flavonoids) and the ecological plasticity of the species, field cultivation or in vitro biomass production of A. mollis are possible alternatives to its collection from nature. Four MS based nutrient media differing in the concentration of the minerals and supplemented with alpha-naphthaleneacetic acid (NAA) and benzylaminopurine (BAP) were tested in order to examine their influence on the shoot multiplication effectiveness and the antioxidant activity of A. mollis, and also the possible relation between these parameters under the conditions of in vitro culture. The cultures grown for two months on these media differed significantly in their multiplication rates (p < 0.001), as well as in their morphological features--height, leaf color and root development. Methanol extracts of in vitro cultivated and ex vitro adapted and acclimated on Vitosha Mt. (1500 m a. s. l.) plants were analyzed for tannin and flavonoid content and for free radical scavenging activity. The contents of flavonoids and tannins in the in vitro cultures of A. mollis cultivated on the four tested media differed significantly (p < 0.05). The highest flavonoid content was found in the shoots cultivated on the control MS medium, as well as in the ex vitro adapted plants. The antioxidant activity of the in vitro cultures correlated positively with the concentrations of the PGRs in the respective media, and the ex vitro adapted plants had the highest antioxidant activity (IC50 13.1 +/- 1.9 microg/mL) commensurable with that of the commercial antioxidant butylated hydroxytoluene (BHT) used as a positive control, with an IC50 of 12.65 microg/mL.     

Intermolecular interactions between the mobile phase components and isomeric naphthols in a thin-layer chromatography

The purpose of our work was to investigate intermolecular interactions between the components of a mobile phase (acetone-benzene) and the isomeric naphthols. On the basis of chromatographic, dielectric, and IR measurements it was established that with the mobile phase composed exclusively of benzene the α- and β-naphthol occurred mainly as monomers and were directly adsorbed on silica gel throughout the whole chromatographic process. With the increase of acetone concentration in the applied mobile phases the effectivity of intermolecular interactions between the α- and β-naphthol molecules and those of acetone also increased. The described method can help give approximate evaluations of intermolecular interactions in the chromatographic systems composed of the mobile phase and separated substances.     

Protein–polymer hybrids: Conducting ARGET ATRP from a genetically encoded cleavable ATRP initiator

Protein–polymer hybrids are an important class of biomaterials. Described is the preparation of a genetically incorporated a non-canonical amino acid (nCAA) containing an ester linked atom transfer radical polymerization (ATRP) initiator, followed by a controlled “grafting from” polymerization. A Methanococcus jannaschii tyrosyl-tRNA synthetase/tRNA_CUA pair was selected to genetically encode p-bromoisobutyryloxymethyl-l-phenylalanine (biF) in response to an amber codon. This biF was directly incorporated into green fluorescent protein (GFP) at residue 134 generating biF-GFP. Activators regenerated by electron transfer (ARGET) ATRP was conducted under biologically relevant conditions to graft well-defined poly(oligo ethylene oxide methacrylate) from the biF-GFP. The biF-GFP retained its biofluorescence properties throughout the polymerization indicating the utility of ARGET ATRP for preparing protein–polymer hybrids. The presence of a base-labile ester bond in the initiator, allowed cleavage of the grafted polymer from the protein and directly analyze their molecular weight and molecular weight distribution using gel permeation chromatography (GPC). The cleaved final polymer had a M_n = 27,000 and a molecular weight distribution of M_w/M_n = 1.27.