Size prediction of recombinant human growth hormone nanoparticles produced by supercritical fluid precipitation

Solution-enhanced dispersion by supercritical fluids (SEDS) was applied to produce nano-sized recombinant human growth hormone (hGH) particles. Ethanol was used to help the supercritical carbon dioxide to extract water from the aqueous protein solution. Various sizes of hGH nanoparticles were successfully prepared with a narrow particle size distribution from aqueous ethanol solution without using any additive. The theoretical particle sizes were deduced from the calculated droplet sizes based on a modified Jasuja’s equation. The calculated mean particle sizes and the experimentally obtained ones were compared and the results showed an excellent correlation coefficient (R ²) of 0.995. Figure Distribution of hGH Nano-particles     

Determination of glucosinolates in traditional Chinese herbs by high-performance liquid chromatography and electrospray ionization mass spectrometry

A reversed-phase HPLC method has been developed for determination of twelve intact glucosinolates—glucoiberin, glucocheirolin, progoitrin, sinigrin, epiprogoitrin, glucoraphenin, sinalbin, gluconapin, glucosibarin, glucotropaeolin, glucoerucin, and gluconasturtiin—in ten traditional Chinese plants. The samples were extracted with methanol and the extracts were cleaned on an activated Florisil column. A mobile phase gradient prepared from methanol and 30 mmol L⁻¹ ammonium acetate at pH 5.0 enabled baseline separation of the glucosinolates. Glucosinolate detection was confirmed by quadrupole time-of-flight tandem mass spectrometric analysis in negative-ionization mode. Detection limits ranged from 0.06 to 0.36 μg g⁻¹ when 5 g of dried plant was analyzed. Recoveries of the glucosinolates were better than 85% and precision (relative standard derivation, n = 3) ranged from 5.3 to 14.6%. Analysis of the glucosinolates provided scientific evidence enabling differentiation of three pairs of easily confused plants. Figure Glucosinolates Analysis for the Differentiation of Easily-Confusing Herbs     

Determining sulfamonomethoxine and its acetyl/hydroxyl metabolites in chicken plasma under organic solvent-free conditions

A quantitative technique is described for a sample preparation followed by high performance liquid chromatography method for the simultaneous determination of sulfamonomethoxine and its metabolites, N ⁴-acetyl SMM and 2,6-dihydroxy SMM, in chicken plasma. The average recoveries, analytical total time, and limits of quantitation were ≥80% (relative standard deviations (SD) ≤6%), <30 min sample-1 (12 samples in 2 h), and ≤0.09 μg ml⁻¹, respectively. The procedure, performed under 100% aqueous conditions, uses no organic solvents and toxic reagents at all and is, therefore, harmless to the environment and humans.      

Elucidation of n-butyl benzyl phthalate biodegradation using high-performance liquid chromatography and gas chromatography–mass spectrometry

n-Butyl benzyl phthalate (BBP) is an endocrine-disrupting chemical. A bacterium species capable of using BBP as the sole source of carbon and energy was isolated from mangrove sediment. Effects of BBP concentration, pH, temperature, and salinity on BBP biodegradation were studied. The optimum pH, temperature, and salinity for the BBP biodegradation were 7.0, 37°C, and 15 g L⁻¹, respectively. BBP was completely degraded within 6 days under optimum conditions, and the biodegradation of BBP could be fitted to a first-order kinetic model. The major metabolites of BBP biodegradation were identified as mono-butyl phthalate, mono-benzyl phthalate, phthalic acid, and benzoic acid by using high-performance liquid chromatography and gas chromatography–mass spectrometry. A preliminary metabolic pathway was proposed for the biodegradation of BBP.      

Colorimetric detection of immunoglobulin G by use of functionalized gold nanoparticles on polyethylenimine film

A method based on use of functionalized gold nanoparticles on polyethylenimine film has been developed for colorimetric detection of immunoglobulin G (IgG). The immunogold nanoparticles were immobilized on quartz slides by recognition between antibody and antigen, with the antigen chemically adsorbed on the polyethylenimine film. By measurement of the UV–visible spectra of the immobilized immunogold, detection of h-IgG was achieved. The detection limit for h-IgG by use of this method can be as low as 0.01 μg mL⁻¹. This method is quite promising for numerous applications in immunoassay. Figure     

Synthesis,characterisation and chromatographic evaluation of polyether dendrimer stationary phases

Supercritical carbon dioxide has attracted attention as a potential replacement for traditional organic solvents due to its simplified workup procedures and reduced environmental impact—providing a green chemistry approach for organic solvent-free functionalisation. In addition to the environmental benefits, the enhanced diffusivity observed in supercritical solvents can often enhance reaction rates. We have applied these valuable features to the preparation of silica-bonded stationary phases and examined their potential in liquid chromatography. We report the successful preparation and characterisation of polyether silica based on Frechet dendrimers—this significantly enhances the range of stationary-phase chemistries that can be prepared in supercritical fluids. First- and second-generation polyether silicas were prepared, characterised, end-capped and evaluated for use as stationary phases for liquid chromatography. Figure SRM1649 on 2nd generation polyether silica Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Highly sensitive spectrofluorimetric determination of trace amounts of lecithin

A new spectrofluorimetric method was developed for the determination of trace amounts of lecithin using the ciprofloxacin (CIP)–terbium (Tb³⁺) ion complex as a fluorescent probe. In a buffer solution at pH=5.60, lecithin can remarkably reduce the fluorescence intensity of the CIP–Tb³⁺ complex at λ=545 nm. The reduced fluorescence intensity of the Tb³⁺ ion is proportional to the concentration of lecithin. Optimum conditions for the determination of lecithin were also investigated. The linear range and detection limit for the determination of lecithin were 1.0×10⁻⁶–3.0×10⁻⁵ mol L⁻¹ and 3.44×10⁻⁷ mol L⁻¹, respectively. This method is simple, practical, and relatively free of interference from coexisting substances. Furthermore, it has been successfully applied to assess lecithin in serum samples.      

Analysis of type I and IV collagens by FT-IR spectroscopy and imaging for a molecular investigation of skeletal muscle connective tissue

Many muscular diseases result from abnormal organization of connective tissue and/or collagen network formation. Only a few molecular imaging techniques are able to analyze this collagen network by differentiating collagen types. In this study, FT-IR spectroscopy was used to analyze type I and IV collagens, the most important compounds of which are perimysium and endomysium, respectively. Secondary structure of collagen types was determined by curve-fitting the 1,700–1,480 cm⁻¹ spectral interval. Type I collagen could be differentiated from type IV by its higher amounts of triple helix and α-helix, but lower amounts of β-sheets (P < 0.01). FT-IR imaging was then used to determine structural features of perimysium and endomysium collagen network in bovine Flexor carpi radialis muscle. Secondary structure of proteins contained in perimysium and endomysium was found to be very close to type I and IV collagens, respectively. FT-IR spectroscopy and imaging are thus analytical tools that might be used for investigating biodistribution and assembly of collagen types in connective tissues. Figure Visible (left) and full spectral FT-IR (right) images of skeletal muscle tissue section (16 μm) exhibiting a vertical arrangement of fibers. + and × in FT-IR image show selected positions to obtain FT-IR spectra of perimysium and endomysium, respectively     

A novel thiocyanate-selective electrode based on a zinc–phthalocyanine complex

A novel thiocyanate (SCN⁻)-selective PVC membrane electrode based on a zinc-phthalocyanine (ZnPc) complex as neutral carrier is described. The membrane electrode containing ZnPc with 5.1% (w/w) ionophore, 29.2% (w/w) PVC, and 65.7% (w/w) 2-nitrophenyl octyl ether (o-NPOE) as plasticizer displayed an anti-Hofmeister selectivity sequence , and exhibited near-Nernstian potential response to thiocyanate ranging from about 1.0×10⁻¹ to 1.0×10⁻⁶ mol L⁻¹ with a detection limit of 7.5×10⁻⁷ mol L⁻¹ and a slope of 58.1±0.5 mV per decade in pH 3.0 phosphate buffer solution at 25 °C. This preferential response is believed to be associated with the unique coordination between the central metal of the carrier and thiocyanate.      

Evaluation of CD detection in an HPLC system for analysis of DHEA and related steroids

The biological importance of dehydroepiandrosterone (DHEA) is reflected by the fact that DHEA is a crucial precursor of the biosynthesis of the steroidal sex hormones. Simultaneous separation of DHEA, dehydroepiandrosterone sulfate (DHEA-S), pregnenolone, androstenedione and testosterone has been accomplished by reversed-phase ion-pair high-performance liquid chromatography (RP-IP-HPLC) based on isocratic elution applying circular dichroism (CD) detection at 295 nm. Addition of tetrabutylammonium hydrogensulfate to the mobile phase increases the retention of DHEA-S on the C8-silica column by an apparent ion-pairing mechanism without affecting the retention of the other (non-ionic) steroids. CD spectroscopy provides highly selective detection of compounds possessing optically active absorption bands and the separation is even more selective in the higher wavelength range applied. The linearity of the steroid concentration (c, mg mL⁻¹) versus peak area was tested in the concentration range of 0.5–2 mg mL⁻¹ (injected quantities were 10–40 μg). The relative standard deviation (RSD) values for DHEA and DHEA-S indicated a good intra-assay and inter-assay precision of the method.      

Determination of trace elements in suspensions and filtrates of drinking and surface water by wavelength-dispersive X-ray fluorescence spectrometry

An X-ray fluorescence method (XRF) is presented that allowed low detection limits (at the 0.1–23 ng mL⁻¹ level) to be obtained for Cr, Mn, Fe, Ni, Zn, Sr, Pb, Bi and Br in water. The samples were prepared using a thin layer method. Trace elements were determined via the calibration curve and standard addition. Absorption effects and inhomogenities in prepared samples were checked for using the emission–transmission method and internal standards, respectively. The results from the XRF method were compared with the results from the inductively coupled plasma atomic emission spectrometry method.      

Screening and analysis of an antineoplastic compound in Rhizoma Chuanxiong by means of in vitro metabolism and HPLC-MS

A new screening and analysis method that combines in vitro metabolism with high-performance liquid chromatography-mass spectrometry (HPLC-MS) was developed for the screening and analysis of an antineoplastic compound, coniferyl ferulate, which is present in the rhizome of Rhizoma Chuanxiong. Infrared (IR), ultraviolet visible spectroscopy (UV-Vis), nuclear magnetic resonance (NMR) and element analysis were used to identify the molecular structure of coniferyl ferulate. The quantitative analysis of coniferyl ferulate in different extracts of Rhizoma Chuanxiong was carried out, and the metabolism of coniferyl ferulate was investigated by in vitro incubation with rat liver homogenate. The metabolite of coniferyl ferulate, ferulic acid ethyl ester, was identified by HPLC-MS, UV-Vis and IR. In addition, antineoplastic activities of coniferyl ferulate and ferulic acid ethyl ester were detected by the MTT assay. The observed inhibition rate of coniferyl ferulate on the activity of HeLa cells was over 80% at 5.4 ng μl⁻¹. However, its metabolite, ferulic acid ethyl ester, showed no antineoplastic activity in vitro.      

Laser ablation inductively coupled plasma mass spectrometry for direct analysis of the spatial distribution of trace elements in metallurgical-grade silicon

The spatial distribution and concentration of impurities in metallurgical-grade silicon (MG-Si) samples (97–99% w/w Si) were investigated by use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The spatial resolution (120 μm) and low limits of detection (mg kg⁻¹) for quality assurance of such materials were studied in detail. The volume-dependent precision and accuracy of non-matrix-matched calibration for quantification of minor elements, using NIST SRM 610 (silicate standard), indicates that LA-ICP-MS is well suited to rapid process control of such materials. Quantitative results from LA-ICP-MS were compared with previously reported literature data obtained by use of ICP-OES and rf-GD-OES. In particular, the distribution of element impurities and their relationship to their different segregation coefficients in silicon is demonstrated. Dedicated to Professor Klaus G. Heumann     

Determination of norfloxacin in human urine by capillary electrophoresis with electrochemiluminescence detection

A fast and sensitive approach that can be used to detect norfloxacin in human urine using capillary electrophoresis with end-column electrochemiluminescence (ECL) detection of is described. The separation column was a 75-μm i.d. capillary. The running buffer was 15 mmol L⁻¹ sodium phosphate (pH 8.2). The solution in the detection cell was 50 mmol L⁻¹ sodium phosphate (pH 8.0) and 5 mmol L⁻¹ The ECL intensity varied linearly with norfloxacin concentration from 0.05 to 10 μmol L⁻¹. The detection limit (S/N=3) was 0.0048 μmol L⁻¹, and the relative standard deviations of the ECL intensity and the migration time for eleven consecutive injections of 1.0 μmol L⁻¹ norfloxacin (n=11) were 2.6% and 0.8%, respectively. The method was successfully applied to the determination of norfloxacin spiked in human urine without sample pretreatment. The recoveries were 92.7–97.9%.      

Preparation of Fe<Subscript>3</Subscript>O<Subscript>4</Subscript>-C18 nano-magnetic composite materials and their cleanup properties for organophosphorous pesticides

Magnetic Fe₃O₄-C18 composite nanoparticles of approximately 5–10 nm in size were synthesized and characterized by IR spectroscopy, atomic absorption spectroscopy, X-ray diffraction, and transmission electron microscopy. The magnetic Fe₃O₄-C18 composite nanoparticles were applied for cleanup and enrichment of organophosphorous pesticides. Comparative studies were carried out between magnetic Fe₃O₄-C18 composite nanoparticles and common C18 materials. Residues of organophosphorous pesticides were determined by gas chromatography in combination with a nitrogen/phosphorus detector. The cleanup and enrichment properties of magnetic Fe₃O₄-C18 composite nanoparticles are comparable with those of common C18 materials for enrichment of organophosphorous pesticides, but the cleanup and enrichment are faster and easier to perform. Figure Presumed mechanism for the adhesion of the OPs to the Fe₃O₄-C18 magnetic nanoparticles     

Nanostructured electrochemical DNA biosensors for detection of the effect of berberine on DNA from cancer cells

Multi walled carbon nanotubes (MWNT) in dimethylformamide (DMF) or aqueous sodium dodecyl sulfate (SDS) solution, colloidal gold nanoparticles (GNP) in phosphate buffer solution (PBS), and a GNP–MWNT mixture in aqueous SDS solution have been investigated for chemical modification of a screen-printed carbon electrode used as the signal transducer of a dsDNA-based biosensor. Differential pulse voltammetry of the DNA redox marker and the guanine moiety anodic oxidation and cyclic voltammetry with K₃[Fe(CN)₆] as indicator revealed substantial enhancement of the response of the biosensor, particularly when MWNT in SDS solution was used. The biosensor was used in testing of berberine, an isoquinoline plant alkaloid with significant antimicrobial and anticancer activity. Berberine had a very strong, concentration-dependent, effect on the structural stability of DNA from the human cancer cells (U937 cells) whereas non-cancer cells were changed only when berberine concentrations were relatively high 75 and 50 μg mL⁻¹. Figure Schematic illustration of preparation of the nanostructured films: (a) layer-to-layer coverage (DNA/nanomaterial/SPE); (b) mixed coverage (DNA-nanomaterial/SPE)     

Preparative HPLC separation of bambuterol enantiomers and stereoselective inhibition of human cholinesterases

We separated and characterized the enantiomers of bambuterol (5-[-(tert-butylamino)-1-hydroxyethyl]-m-phenylene-bis(dimethylcarbamate) hydrochloride), which is used in racemic form as a prodrug of terbutaline, a β₂-adrenoceptor agonist. The enantioseparation was attempted on several chiral HPLC columns, and the most effective separation was achieved on the amylose-based Chiralpak AD column. Since in vivo conversion of bambuterol into terbutaline involves hydrolysis by butyrylcholinesterase (EC 3.1.1.8), we studied the reaction of enantiomers with eight human BChE variants. Both enantiomers inhibited all studied BChE variants; however, the rate of inhibition with the (R)-enantiomer was about five times faster than with the (S)-enantiomer. (R)-bambuterol inhibition rate constants for homozygous usual (UU), fluoride-resistant (FF) or atypical (AA) variant ranged from 6.4 to 0.11 min^-1μM^-1. The inhibition rates for heterozygotes were between the respective constants for the corresponding homozygotes. An erratum to this article can be found at     

Investigating the post-chemiluminescence behavior of phenothiazine medications in the luminol–potassium ferricyanide system: molecular imprinting–post-chemiluminescence method for the determination of chlorpromazine hydrochloride

A new post-chemiluminescence (PCL) phenomenon was observed when phenothiazine medications were injected into the reaction mixture after the chemiluminescence (CL) reaction of luminol and potassium ferricyanide had finished. A possible reaction mechanism was proposed based on studies of the kinetic characteristics of the CL, CL spectra, fluorescence spectra, and on other experiments. The feasibility of determining various phenothiazine medications by utilizing these PCL reactions was examined. A molecular imprinting–post-chemiluminescence (MI-PCL) method was established for the determination of chlorpromazine hydrochloride using a chlorpromazine hydrochloride-imprinted polymer (MIP) as the recognition material. The method displayed high selectivity and high sensitivity. The linear range of the method was 1.0×10⁻⁸∼1.0×10⁻⁶, with a linear correlation coefficient of 0.9985. The detection limit was 3×10⁻⁹ g/ml chlorpromazine hydrochloride, and the relative standard deviation for a 1.0×10⁻⁷ g/ml chlorpromazine hydrochloride solution was 4.0% (n=11). The method has been applied to the determination of chlorpromazine hydrochloride in urine and animal drinking water with satisfactory results.      

Towards an unbiased metabolic profiling of protozoan parasites: optimisation of a Leishmania sampling protocol for HILIC-orbitrap analysis

Comparative metabolomics of Leishmania species requires the simultaneous identification and quantification of a large number of intracellular metabolites. Here, we describe the optimisation of a comprehensive metabolite extraction protocol for Leishmania parasites and the subsequent optimisation of the analytical approach, consisting of hydrophilic interaction liquid chromatography coupled to LTQ-orbitrap mass spectrometry. The final optimised protocol starts with a rapid quenching of parasite cells to 0 °C, followed by a triplicate washing step in phosphate-buffered saline. The intracellular metabolome of 4 × 10⁷ parasites is then extracted in cold chloroform/methanol/water 20/60/20 (v/v/v) for 1 h at 4 °C, resulting in both cell disruption and comprehensive metabolite dissolution. Our developed metabolomics platform can detect approximately 20% of the predicted Leishmania metabolome in a single experiment in positive and negative ionisation mode.