Quantitative determination of vitamin D metabolites in plasma using UHPLC-MS/MS

Vitamin D is an important determinant of bone health at all ages. The plasma concentrations of 25-hydroxy vitamin D (25-OH D) and other metabolites are used as biomarkers for vitamin sufficiency and function. To allow for the simultaneous determination of five vitamin D metabolites, 25-OH D₃, 25-OH D₂, 24,25-(OH)₂ D₃, 1,25-(OH)₂ D₃, and 1,25-(OH)₂ D₂, in low volumes of human plasma, an assay using ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) was established. Plasma samples were spiked with isotope-labeled internal standards and pretreated using protein precipitation, solid-phase extraction (SPE) and a Diels–Alder derivatization step with 4-phenyl-1,2,4-triazoline-3,5-dione. The SPE recovery rates ranged from 55% to 85%, depending on the vitamin D metabolite; the total sample run time was <5 min. Mass spectrometry was conducted using positive ion electrospray ionization in the multiple reaction monitoring mode on a quadrupole–quadrupole-linear ion trap instrument after pre-column addition of methylamine to increase the ionization efficiency. The intra- and inter-day relative standard deviations were 1.6–4.1% and 3.7–6.8%, respectively. The limit of quantitation for these compounds was determined to be between 10 and 20 pg/mL. The 25-OH D results were compared with values obtained for reference materials (DEQAS). In addition, plasma samples were analyzed with two additional Diasorin antibody assays. All comparisons with conventional methods showed excellent correlations (r ² = 0.9738) for DEQAS samples, demonstrating the high degree of comparability of the new UHPLC-MS/MS technique to existing methods.     

Unsymmetrically substituted N-heterocyclic phosphenium ions

Reduction of an unsymmetrically substituted α-diimine followed by condensation with PCl₃ yielded a P-chloro-N-aryl-N′-alkyl diazaphospholene which was further converted into an unsymmetrical diazaphospholium triflate by reaction with trimethylsilyl triflate. Reaction of tetramers of N-H- or N-alkyl-benzo-1,3,2-diazaphospholes with methyl triflate or triflic acid led in one step to triflate salts of unsymmetrically substituted benzo-1,3,2-diazaphospholium cations. Determination of the crystal structures of two of these derivatives by single-crystal X-ray diffraction studies revealed that individual cations and anions in the crystal lattice interact via specific electrostatic, π-stacking, or van-der-Waals type interactions to form supramolecular assemblies. Thermoanalytical measurements disclosed that benzo-diazaphospholium triflates with medium length alkyl chains melt below 100 °C and exhibit a strong tendency to form supercooled liquids.     

Phosphinidene generation from phosphorus heterocycles and cages – A theoretical study

The dissociation properties of several phosphorus heterocycles and cages (complexes of phosphinidenes with P-donor ligands) were investigated using DFT methods. Good correlations have been found between the dissociation energies of the complexes with P and N ligands. The comparison with N-donor complexes revealed that the stability of complexes with pyrido-annelation(s) is very similar. The trends in dissociation energies for the benzannelated complexes are identical; however, the complexes with P ligands are much more stable than those with N ligands. The major reason for the different behavior is the pyramidality of the bridgehead P/N atoms in these complexes.     

CRISPR/Cas9-Mediated Genome Editing in Comfrey (Symphytum officinale) Hairy Roots Results in the Complete Eradication of Pyrrolizidine Alkaloids

Comfrey (Symphytum officinale) is a medicinal plant with anti-inflammatory, analgesic, and proliferative properties. However, its pharmaceutical application is hampered by the co-occurrence of toxic pyrrolizidine alkaloids (PAs) in its tissues. Using a CRISPR/Cas9-based approach, we introduced detrimental mutations into the hss gene encoding homospermidine synthase (HSS), the first pathway-specific enzyme of PA biosynthesis. The resulting hairy root (HR) lines were analyzed for the type of gene-editing effect that they exhibited and for their homospermidine and PA content. Inactivation of only one of the two hss alleles resulted in HRs with significantly reduced levels of homospermidine and PAs, whereas no alkaloids were detectable in HRs with two inactivated hss alleles. PAs were detectable once again after the HSS-deficient HRs were fed homospermidine confirming that the inability of these roots to produce PAs was only attributable to the inactivated HSS and not to any unidentified off-target effect of the CRISPR/Cas9 approach. Further analyses showed that PA-free HRs possessed, at least in traces, detectable amounts of homospermidine, and that the PA patterns of manipulated HRs were different from those of control lines. These observations are discussed with regard to the potential use of such a CRISPR/Cas9-mediated approach for the economical exploitation of in vitro systems in a medicinal plant and for further studies of PA biosynthesis in non-model plants.     

Effects of zeolites on volatile products of beech wood using analytical pyrolysis

Five unmodified basic and acidic zeolites have been tested in the thermo-chemical conversion of beech (Fagus sylvatica L.) using an on-line pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) system to study their effects on lignocellulose thermal degradation products. The catalysts employed were SN-27, MSN-15, MSM-15, H-ZSM-5-28 and H-ZSM-5-80 with varying Si/Al ratio between 11 and 80. Thoroughly mixed samples of the biomass with different catalysts loading were pyrolysed at 500 °C. The catalyst exhibited varying degree of selectivity on individual volatile products. The selectivity was largely controlled by the nature of zeolites’ active sites and alumina contents. The acidic catalysts mostly influenced the condensable organic products; induced high yields of furfural and levoglucosan. Their yields increased up to five and nine times respectively in samples spiked with 40% MSM-15 and H-ZMS-5 on the basis of feedstock weight. H-ZSM-5 with lower alumina content proved most effective in enhancing high yield of furfural due to better diffusion of molecules through the pores. A slight reduction in polysaccharide derived low molecular volatiles such as hydroxyacetaldehyde, acetic acid, hydroxypropanone was observed in all catalysts. The overall result indicated that the acidic catalysts largely induced cleavage of glycosidic bonds through protonation predominantly on zeolites’ surface area. This was followed by the modification of free hydroxyl groups through dehydration process. All zeolites revealed no significant effect on most lignin derived products except indenes.     

Stereoselective synthesis of both enantiomers of rugulactone

The stereoselective total synthesis of both enantiomers of rugulactone 1 has been completed by applying enantioselective allyl additions as key steps. Two different strategies based on highly stable and enantiomerically pure α-substituted allylboronic esters 2 and 3 were performed starting from boronic ester 4.     

Amidoamine-based dendrimers with end-grafted Pd-Fe units: Synthesis, characterization and their use in the Heck reaction

The synthesis and characterization of novel amidoamine-based metallodendrimers with heterobimetallic end-grafted amidoferrocenyl-palladium-allyl chloride units is described. Dendrimer (Fe((η⁵-C₅H₄PPh₂)(η⁵-C₅H₄))C(O)HNCH₂CH₂NHC(O)CH₂CH₂)N[CH₂CH₂N(CH₂CH₂C(O)NHCH₂CH₂NH-C(O)(Fe(η⁵-C₅H₄)(η⁵-C₅H₄PPh₂)))₂]₂ (9-Fe) and the corresponding metal species (Fe((η⁵-C₅H₄PPh₂(Pd(η³-C₃H₅)Cl))(η⁵-C₅H₄))C(O)HNCH₂CH₂NHC(O)CH₂CH₂)N[CH₂CH₂N(CH₂CH₂C(O)NHCH₂CH₂NHC(O)(Fe(η⁵-C₅H₄)(η⁵-C₅H₄PPh₂(Pd(η³-C₃H₅)Cl))))₂]₂ (9-Fe-Pd) were prepared by a consecutive divergent synthesis methodology including addition-amidation cycles, standard peptide coupling, and coordination procedures. For comparative reasons also the monomeric and dimeric molecules (Fe(η⁵-C₅H₄PPh₂)(η⁵-C₅H₄C(O)NHⁿC₃H₇)) (5-Fe) and [Fe(η⁵-C₅H₄PPh₂)(η⁵-C₅H₄C(O)NHCH₂)]₂ (6-Fe) as well as N(CH₂CH₂C(O)NHCH₂CH₂NHC(O)(Fe(η⁵-C₅H₄)(η⁵-C₅H₄PPh₂)))₃ (7-Fe) and [CH₂N(CH₂CH₂C(O)NHCH₂CH₂NHC(O)(Fe(η⁵-C₅H₄)(η⁵-C₅H₄PPh₂)))₂]₂ (8-Fe) were prepared from Fe(η⁵-C₅H₄PPh₂)(η⁵-C₅H₄CO₂H) (3). Using [Pd(η³-C₃H₅)Cl]₂ (4) as palladium source heterobimetallic metallodendrimers (Fe(η⁵-C₅H₄PPh₂(Pd(η³-C₃H₅)Cl))(η⁵-C₅H₄C(O)NHⁿC₃H₇)) (5-Fe-Pd), [Fe(η⁵-C₅H₄PPh₂(Pd(η³-C₃H₅)Cl))(η⁵-C₅H₄C(O)NHCH₂)]₂ (6-Fe-Pd), N(CH₂CH₂C(O)NHCH₂CH₂NHC(O)(Fe(η⁵-C₅H₄)(η⁵-C₅H₄PPh₂(Pd(η³-C₃H₅)Cl))))₃ (7-Fe-Pd) and [CH₂N(CH₂CH₂C(O)NHCH₂CH₂NHC(O)(Fe(η⁵-C₅H₄)(η⁵-C₅H₄PPh₂(Pd(η³-C₃H₅)Cl))))₂]₂ (8-Fe-Pd) were synthesized. Additionally, seleno-phosphines of 5-Fe-Se and 9-Fe-Se, respectively, were prepared by addition of elemental selenium to 5-Fe or 9-Fe to estimate their σ-donor properties.The palladium-containing amidoamine supports are catalytically active in the Heck-Mizoroki cross-coupling of iodobenzene with tert-butyl acrylate. The catalytic data are compared to those obtained for the appropriate mononuclear and dinuclear compounds 5-Fe-Pd and 6-Fe-Pd. This comparison confirms a positive cooperative effect. The mercury drop test showed that (nano)particles were formed during catalysis, following on heterogeneous carbon-carbon cross-coupling.     

High performance thin layer chromatography determination of cellobiosan and levoglucosan in bio-oil obtained by fast pyrolysis of sawdust

In this work, high performance thin layer liquid chromatography (HTPLC) is applied to the determination of sugars in fast pyrolysis liquids (bio-oil) and fractions thereof. The proposed procedure allows the separation of anhydrosugar levoglucosan and cellobiosan, as well as glucose, arabinose, xylose and cellobiose. Pre-treatment and derivatization of samples are not necessary and volatile compounds present in bio-oil do not interfere with sugar analysis. The detrimental effect of the complex bio-oil matrix on columns and detector lifetime is avoided by using disposable HTPLC plates. Prior screening of glucose, present especially in aged and aqueous bio-oil fractions, is required to quantify cellobiosan without interference. Concentrations of levoglucosan and cellobiosan in bio-oil samples obtained from Pinus radiata sawdust were ranged between 1.27-2.26% and 0.98-1.96% respectively, while a bio-oil sample obtained from native wood contained a higher levoglucosan concentration.