Evaluation of hydrophilic interaction chromatography versus reversed-phase chromatography for fast aqueous species distribution analysis of Nickel(II)-Histidine complex species.

Nickel (Ni) is a trace heavy metal of importance in biological and environmental systems, with well documented allergy and carcinogenic effects in humans. With Ni(II) as the dominant oxidation state, the elucidation of the coordination mechanisms and labile complex species responsible for its transportation, toxicity, allergy, and bioavailability is key to understanding its biological effects and location in living systems. Histidine (His) is an essential amino acid that contributes to protein structure and activity and in the coordination of Cu(II) and Ni(II) ions. The aqueous low molecular weight Ni(II)-Histidine complex consists primarily of two stepwise complex species Ni(II)(His)1 and Ni(II)(His)2 in the pH range of 4 to 12. Four chromatographic columns, including the superficially porous Poro-shell EC-C18, Halo RP-amide and Poro-shell bare silica-HILIC columns, alongside a Zic-cHILIC fully porous column, were evaluated for the fast separation of the individual Ni(II)-Histidine species. Of these the Zic-cHILIC exhibited high efficiency and selectivity to distinguish between the two stepwise species Ni(II)His1 and Ni(II)His2 as well as free Histidine, with a fast separation within 120 s at a flow rate of 1 ml/min. This HILIC method utilizing the Zic-cHILIC column was initially optimized for the simultaneous analysis of Ni(II)-His-species using UV detection with a mobile phase consisting of 70% ACN and sodium acetate buffer at wwpH 6. Furthermore, the aqueous metal complex species distribution analysis for the low molecular weight Ni(II)-histidine system was chromatographically determined at various metal-ligand ratios and as a function of pH. The identities of Ni(II)His1 and Ni(II)-His2 species were confirmed using HILIC electrospray ionization- mass spectrometry (HILIC-ESI-MS) at negative mode.

Isoquinolinequinone N-oxides as anticancer agents effective against drug resistant cell lines.

The isoquinolinequinone (IQQ) pharmacophore is a privileged framework in known cytotoxic natural product families, caulibugulones and mansouramycins. Exploiting both families as a chemical starting point, we report on the structured development of an IQQ N-oxide anticancer framework which exhibits growth inhibition in the nM range across melanoma, ovarian and leukaemia cancer cell lines. A new lead compound (16, R6 = benzyl, R7 = H) exhibits nM GI50 values against 31/57 human tumour cell lines screened as part of the NCI60 panel and shows activity against doxorubicin resistant tumour cell lines. An electrochemical study highlights a correlation between electropositivity of the IQQ N-oxide framework and cytotoxicity. Adduct binding to sulfur based biological nucleophiles glutathione and cysteine was observed in vitro. This new framework possesses significant anticancer potential.

Biotin interference in immunoassays based on biotin-strept(avidin) chemistry: An emerging threat.

Biotinylated antibodies/antigens are currently used in many immunoassay formats in clinical settings for diversified analytes and biomarkers to offer high detection selectivity and sensitivity. Biotin cannot be synthesized by mammals and must be taken as an essential supplement. Normal intake of biotin from various foods and milk causes no effect on the streptavidin/biotin-based immunoassays. However, overconsumption of biotin (daily doses 100-300 mg) poses a significant problem for immunoassays using the biotin-strept(avidin) pair. Biotin interferences are noted in immunoassays of thyroid markers, drugs, hormones, cancer markers, the biomarker for cardiac function (ß-human chorionic gonadotropin), etc. The biotin level required for serious interference in test results varies significantly from test to test and cannot easily be predicted. Immunoassay manufacturers with technologies based on strept(avidin)-biotin binding must investigate the interference from biotin (up to at least 1200 ng/mL or 4.9 µM of biotin) in various formats. There is no concrete solution to circumvent the biotin interference encountered in blood samples, short of biotin removal. Considering the short half-life of biotin in the human body, patients must stop taking biotin supplements for >48 h before the test. However, this scenario is not considered for patients in emergency situations or those with biotinidase deficiency, mitochondrial metabolic disorders or multiple sclerosis. Apparently, a rapid analytical procedure for biotin is urgently needed to quantify for its interference in immunoassays using strep(avidin)-biotin chemistry. To date, there is no quick and reliable procedure for the detection of biotin at below nanomolar levels in blood and biological samples. Traditional lab-based techniques including HPLC/MS-MS cannot process an enormous number of public samples. Biosensors with high detection sensitivity, miniaturization, low cost, and multiplexing have the potential to address this issue.

Rapid Electrochemical Detection of Pseudomonas aeruginosa Signaling Molecules by Boron-Doped Diamond Electrode.

As the leading cause of morbidity and mortality of cystic fibrosis (CF) patients, early detection of Pseudomonas aeruginosa (PA) is critical in the clinical management of this pathogen. Herein, we describe rapid and sensitive electroanalytical methods using differential pulse voltammetry (DPV) at a boron-doped diamond (BDD) electrode for the detection of PA signaling biomolecules. Monitoring the production of key signaling molecules in bacterial cultures of P. aeruginosa PA14 over 8 h is described, involving sample pretreatment by liquid-liquid and solid-phase extraction. In addition, direct electrochemical detection approach of PA signaling molecules is also reported in conjunction with hexadecyltrimethylammonium bromide (CTAB) to disrupt the bacterial membrane.

Synthesis and electrochemical detection of a thiazolyl-indole natural product isolated from the nosocomial pathogen Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen, capable of surviving in a broad range of natural environments and quickly acquiring resistance. It is associated with hospital-acquired infections, particularly in patients with compromised immunity, and is the primary cause of morbidity and mortality in cystic fibrosis (CF) patients. P. aeruginosa is also of nosocomial importance on dairy farms and veterinary hospitals, where it is a key morbidity factor in bovine mastitis. P. aeruginosa uses a cell-cell communication system consisting of signalling molecules to coordinate bacterial secondary metabolites, biofilm formation, and virulence. Simple and sensitive methods for the detection of biomolecules as indicators of P. aeruginosa infection would be of great clinical importance. Here, we report the synthesis of the P. aeruginosa natural product, barakacin, which was recently isolated from the bovine ruminal strain ZIO. A simple and sensitive electrochemical method was used for barakacin detection using a boron-doped diamond (BDD) and glassy carbon (GC) electrodes, based on cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The influence of electrolyte pH on the peak potential and peak currents was also investigated. At pH 2.0, the peak current was linearly dependent on barakacin concentration (in the range used, 1-10 µM), with correlation coefficients greater than 0.98 on both electrodes. The detection limit (S/N = 3) on the BDD electrode was 100-fold lower than that obtained on the GC electrode. The optimized method using the BDD electrode was extended to bovine (cow feces) and human (sputum of a CF patient) samples. Spiked barakacin was easily detected in these matrices at a limit of 0.5 and 0.05 µM, respectively. Graphical abstract Electrochemical detection of barakacin.

Molecular Signature of Pseudomonas aeruginosa with Simultaneous Nanomolar Detection of Quorum Sensing Signaling Molecules at a Boron-Doped Diamond Electrode.

Electroanalysis was performed using a boron-doped diamond (BDD) electrode for the simultaneous detection of 2-heptyl-3-hydroxy-4-quinolone (PQS), 2-heptyl-4-hydroxyquinoline (HHQ) and pyocyanin (PYO). PQS and its precursor HHQ are two important signal molecules produced by Pseudomonas aeruginosa, while PYO is a redox active toxin involved in virulence and pathogenesis. This Gram-negative and opportunistic human pathogen is associated with a hospital-acquired infection particularly in patients with compromised immunity and is the primary cause of morbidity and mortality in cystic fibrosis (CF) patients. Early detection is crucial in the clinical management of this pathogen, with established infections entering a biofilm lifestyle that is refractory to conventional antibiotic therapies. Herein, a detection procedure was optimized and proven for the simultaneous detection of PYO, HHQ and PQS in standard mixtures, biological samples, and P. aeruginosa spiked CF sputum samples with remarkable sensitivity, down to nanomolar levels. Differential pulse voltammetry (DPV) scans were also applicable for monitoring the production of PYO, HHQ and PQS in P. aeruginosa PA14 over 8 h of cultivation. The simultaneous detection of these three compounds represents a molecular signature specific to this pathogen.

Retention and selectivity properties of carbamate pesticides on novel polar-embedded stationary phases.

This study describes the use of stationary phases with polar functionality suitable for the chemical analysis of carbamates pesticides and comparing with conventional alkyl C8 and C18 phases. The emphasis of this study was to compare the selectivity and retention of the pesticides on different stationary phases, bonded onto 1.7 microm partially porous silica particles under isocratic separation condition. Four stationary phases including: phenylaminopropyl (PAP) phase, bidentate propylurea-C18 (BPUC(18)), C8 and C18, were successfully bonded on the partially porous silica spheres as evidenced by (29)Si and (13)C solid-state NMR analysis. The phenylaminopropyl phase exhibited smaller retentivity and enhanced selectivity compared to the alkyl C8 phase; the analysis time to run separation of the six carbamate pesticides (i.e., methomyl, propoxur, carbofuran, carbaryl, isoprocarb, and promecarb) on the PAP phase was threefold faster than alkyl C8 phase. In a similar manner, the BPUC(18) phase shows similar selectivity to that of the PAP phase, but with longer retentivity; although the BPUC(18) phase is characterized with a lesser degree of retentivity for the carbamate pesticides than the conventional alkyl C18 phase. We propose that pi-pi and weak polar interactions between the carbamate pesticides and the PAP phase dominates the separation mechanism and providing a superior selectivity; faster separation time was also achieved as a result of smaller retentivity. Whereas the C8 and C18 bonded phases exhibits only hydrophobic interactions with the pesticides, leading to larger retentivity. The BPUC(18) phase is shown to interact via polar-polar interactions in addition to hydrophobic interactions with the pesticides, providing similar selectivity with the PAP phase but with larger retentivity.

Cyclodextrin-modified capillary electrophoresis for achiral and chiral separation of ergostane and lanostane compounds extracted from the fruiting body of Antrodia camphorata.

A CD-modified capillary electrophoretic method has been developed for achiral and chiral analysis of seven bioactive compounds isolated from the fruiting body of Antrodia camphorata. Such important target analytes exhibit similar chemical structures and are known for their diverse properties including antioxidant and anticancer effects. The analytes were separated in 25 min using a pH 9.3, 20 mM sodium borate buffer containing 20 mM methyl-beta-CD and 30 mM sulfobutylether-beta-CD. With the exception of the optical isomer pairs (antcin B or zhankuic acid A, zhankuic acid C, and antcin A), the remaining bioactive compounds including the chiral pair antcin C were baseline-separated. Analysis time was noticeably longer to baseline separate all of the above chiral pairs (approximately 38 min) by adding 5% DMF to the running buffer. The migration order was reversed compared with the HPLC elution. More hydrophobic compounds complexed favorably with methyl-beta-CD and emerged earlier in the electropherogram than their more hydrophilic counterparts which were strongly associated with sulfobutylether-beta-CD. The simple capillary electrophoretic method developed was applicable for rapid separation and characterization of several important bioactive compounds isolated from the fruiting body of A. camphorata.

Selective nanomolar detection of dopamine using a boron-doped diamond electrode modified with an electropolymerized sulfobutylether-beta-cyclodextrin-doped poly(N-acetyltyramine) and polypyrrole composite film.

N-acetyltyramine was synthesized and electropolymerized together with a negatively charged sulfobutylether-beta-cyclodextrin on a boron-doped diamond (BDD) electrode followed by the electropolymerization of pyrrole to form a stable and permselective film for selective dopamine detection. The selectivity and sensitivity of the formed layer-by-layer film was governed by the sequence of deposition and the applied potential. Raman results showed a decrease in the peak intensity at 1329 cm(-1) (sp(3)), the main feature of BDD, upon each electrodeposition step. Such a decrease was correlated well with the change of the charge-transfer resistance derived from impedance data, i.e., reflecting the formation of the layer-by-layer film. The polycrystalline BDD surface became more even with lower surface roughness as revealed by scanning electron and atomic force microscopy. The modified BDD electrode exhibited rapid response to dopamine within 1.5-2 s and a low detection limit of 4-5 nM with excellent reproducibility. Electroactive interferences caused by 4-dihydroxyphenylalanine, 3,4-dihydroxyphenylacetic acid, ascorbic acid, and uric acid were completely eliminated, whereas the signal response of epinephrine and norepinephrine was significantly suppressed by the permselective film.