An innovative monolithic zwitterionic stationary phase for the separation of phenolic acids in coffee bean extracts by capillary electrochromatography.

A methacrylate based monolith, containing the innovative zwitterionic monomer (3-allyl-1-imidazol)propane sulfonate, was prepared in 100 µm I.D. silica capillaries by UV initiated photo-polymerization. Composition of the porogen, i.e. a mixture of 1-propanol, 1,4 butanediol and water, was of great importance to obtain a homogeneous monolith with satisfactory permeability and good electrochromatographic performance. Morphology of the stationary phase was studied in Scanning Electron Microscopy and IR experiments, which revealed a good attachment to the capillary wall, flowthrough-pores in the range of 0.5-2 µm, and a continuous monolithic structure. The developed material was well suited for the analysis of six common phenolic acids (salicylic, cinnamic, syringic, rosmarinic, caffeic and chlorogenic acid) by CEC. Their separation was possible in less than 8 min with a mobile phase comprising a 12 mM aqueous ammonium acetate solution with pH 8.5 and acetonitrile, at an applied voltage of - 20 kV. The developed method was validated (R2 ≥ 0.995; LOD ≤ 3.9 µg mL-1, except for salicylic acid; recovery rates from 94 to 104%) and successfully used for the determination of phenolic acids in Coffea arabica samples. All of them contained cinnamic, syringic and caffeic acid, however only in unroasted coffee beans chlorogenic acid (0.06%) was found. The quantitative results were in good agreement to reported literature data.

Hepatic but not brain iron is rapidly chelated by deferasirox in aceruloplasminemia due to a novel gene mutation.

BACKGROUND & AIMS: Aceruloplasminemia is a rare autosomal recessive neurodegenerative disease associated with brain and liver iron accumulation which typically presents with movement disorders, retinal degeneration, and diabetes mellitus. Ceruloplasmin is a multi-copper ferroxidase that is secreted into plasma and facilitates cellular iron export and iron binding to transferrin. RESULTS: A novel homozygous ceruloplasmin gene mutation, c.2554+1G>T, was identified as the cause of aceruloplasminemia in three affected siblings. Two siblings presented with movement disorders and diabetes. Complementary DNA sequencing showed that this mutation causes skipping of exon 14 and deletion of amino acids 809-852 while preserving the open reading frame. Western blotting of liver extracts and sera of affected patients showed retention of the abnormal protein in the liver. Aceruloplasminemia was associated with severe brain and liver iron overload, where hepatic mRNA expression of the iron hormone hepcidin was increased, corresponding to the degree of iron overload. Hepatic iron concentration normalized after 3 and 5months of iron chelation therapy with deferasirox, which was also associated with reduced insulin demands. During short term treatment there was no clinical or imaging evidence for significant effects on brain iron overload. CONCLUSIONS: Aceruloplasminemia can show an incomplete clinical penetrance but is invariably associated with iron accumulation in the liver and in the brain. Iron accumulation in aceruloplasminemia is a result of defective cellular iron export, where hepcidin regulation is appropriate for the degree of iron overload. Iron chelation with deferasirox was effective in mobilizing hepatic iron but has no effect on brain iron.

Quantification of low-density and high-density lipoproteins in human serum by material enhanced infrared spectroscopy (MEIRS).

A key risk factor in the development of atherosclerosis is a high concentration of serum low density lipoprotein (LDL)-cholesterol. The main purpose of this study was to assess the LDL and high density lipoprotein (HDL) content in human serum by employing near-infrared (NIR) spectroscopy and multivariate calibration techniques. Initially a qualitative principal component analysis (PCA) based cluster model was generated to evaluate the feasibility of NIRS for classifying and identifying the LDL and HDL-cholesterol. Therefore TiO(2) beads were used as an adsorbent for selectively immobilizing LDL and HDL-cholesterol and further analysing the incubated and washed samples via NIR diffuse reflection spectroscopy. A principle component regression (PCR) model of 24 LDL standards in a range from 500 - 3000 ppm (clinical value is 1500 ppm) and a partial least squares regression (PLSR) model of 25 HDL standards in a range from 100 - 1000 ppm (clinical value is 400 ppm) were computed. Furthermore, the wavenumber region between 4000 cm(-1) and 7240 cm(-1) was found comprising the main spectral information regarding the TiO(2)-LDL and TiO(2)-HDL composites. The regression coefficients (r) for LDL and HDL were > 0.99 (calibration curve) and > 0.97 (validation curve), respectively. The PCR model of TiO(2)-LDL showed a standard error of estimation (SEE) of 122.80 ppm and a standard error of prediction (SEP) of 121.15 ppm while the PLSR model of TiO(2)-HDL showed 47.70 and 47.14 ppm, respectively. In order to determine the concentration of HDL in real serum samples, LDL was removed by adding a precipitation reagent containing 10 mg/mL magnesium dextran-sulfate, followed by incubation and centrifugation. The pretreated serum samples were predicted by the PLSR model while the standard deviation (SD) from the reference to the NIR predicted values of six test samples in a concentration range from 500 - 2500 ppm showed < 10 %. These results indicate the usefulness of the NIR spectroscopy (NIRS) as a potential alternative or even supplementary clinical method for the quick determination of LDL and HDL in human serum.

Identification of carbohydrates by matrix-free material-enhanced laser desorption/ionisation mass spectrometry.

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) is a sensitive mass spectrometric technique which utilises acidic materials as matrices for laser energy absorption, desorption and ionisation of analytes. These matrix materials produce background signals particularly in the low-mass range and make the detection and identification of small molecules difficult and nearly impossible. To overcome this problem this paper introduces matrix-free material-enhanced laser desorption/ionisation mass spectrometry (mf-MELDI-MS) for the screening and analysis of small molecules such as carbohydrates. For this purpose, 4,4'-azo-dianiline was immobilised on silica gel enabling the absorption of laser energy sufficient for successful desorption and ionisation of low molecular weight compounds. The particle and pore sizes, the solvent system for suspension and the sample preparation procedures have been optimised. The newly synthesised MELDI material delivered excellent spectra with regard to signal-to-noise ratio and detection sensitivity. Finally, wheat straw degradation products and Salix alba L. plant extracts were analysed proving the high performance and excellent behaviour of the introduced material.

Analysis of flavonoids by CE using capacitively coupled contactless conductivity detection.

A CE method employing capacitively coupled contactless conductivity (C(4)D) compared to indirect UV-detection was developed for the analysis of phytochemically relevant flavonoids, such as 6-hydroxyflavone, biochanin A, hesperetin and naringenin. To ensure fast separation at highest selectivity, sensitivity and peak symmetry, the pH value and the concentration of the running BGE had to be optimized regarding both co- and counter-EOF mode. Optimum conditions were found to be 1.0 and 5.0 mM chromate BGE (pH 9.50) in the counter- and co-EOF mode, respectively. Validation of the established CE-C(4)D method pointed out to be approximately seven times more sensitive compared to indirect UV-detection applying the same conditions. The lower LOD defined at an S/N of 3:1 was found between 0.12 and 0.21 microg/mL for the analytes of interest using C(4)D and between 0.77 and 1.20 microg/mL using indirect UV-detection. Compared to an earlier published CE method employing direct UV-detection, C(4)D was found to be approximately two times more sensitive. Due to the lower baseline noise, C(4)D showed an excellent regression coefficient >0.99 compared to 0.93 when using indirect UV detection calibrating within a concentration range between 1 and 10 microg/mL. The influence of the sugar moiety on the conductivity of a flavonoid was studied upon the analysis of the aglycon hesperetin and the rutinosid hesperidin. The sugar moiety in hesperedin shows a higher conductivity compared to hesperetin. Finally, the optimized established CE-C(4)D method was applied to the determination and quantification of naringenin in Sinupret.