Degradation effects in the extraction of antioxidants from birch bark using water at elevated temperature and pressure

Experiments with birch bark samples have been carried to enable a distinction between extraction and degradation effects during pressurised hot water extraction. Two samples, E80 and E180, contained birch bark extracts obtained after extraction at 80 and 180 °C for up to 45 min, respectively. Two other samples, P80 and P180, were only extracted for 5 min at the two temperatures and were thereafter filtered and hydrothermally treated at 80 and 180 °C, respectively. During the latter treatment, samples were collected at different times to assess the stability of the extracted compounds. An offline DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, as well as a high performance liquid chromatographic separation coupled to an electrochemical detector, were used to determine the antioxidant capacity of the processed samples. The results obtained with the different techniques were compared to assess the yield of the extraction and degradation processes. In addition, an online hyphenated system comprising high performance liquid chromatography coupled to diode-array; electrochemical; and tandem mass spectrometric detection (HPLC-DAD-ECD-MS/MS) was used to study the compositions of the extracts in more detail. The results for the samples processed at 80 °C showed that the extraction reached a steady-state already after 5 min, and that the extracted compounds were stable throughout the entire extraction process. Processing at 180 °C, on the other hand, gave rise to partly degraded extracts with a multitude of peaks in both the diode array and electrochemical detectors, and a higher antioxidant capacity compared to for the extracts obtained at 80 °C. It is concluded that HPLC-DAD-ECD is a more appropriate technique for the determination of antioxidants than the DPPH assay. The mass spectrometric results indicate that one of the extracted antioxidants, catechin, was isomerised to its diastereoisomers; (+)-catechin, (−)-catechin, (+)-epicatechin, and (−)-epicatechin.     

MAOS ls for the general synthesis and lead optimization of 3,6-disubstituted-[1,2,4]triazolo[4,3-b]pyridazines

General, high-yielding MAOS protocols for the expedient synthesis of functionalized 3,6-disubstituted-[1,2,4]triazolo[4,3-b]pyridazines are described amenable to an iterative analog library synthesis strategy for the lead optimization of an M1 antagonist screening hit. Optimized compounds proved to be highly selective M1 antagonists.     

Gas-phase acidity, bond dissociation energy and enthalpy of formation of fluorine-substituted benzenes: A theoretical study

A variety of theoretical methods have been used to study the gas-phase acidity of benzene and its eleven fluorine-substituted derivatives: fluorobenzene, three isomers of difluorobenezene, three isomers of trifluorobenzene, three isomers of tetrafluorobenzene and 1,2,3,4,5-pentafluorobenzene. The high-level ab initio methods, G3//B3-LYP and CBS-QB3, are shown to reproduce experimental data to within an average of 1.9 and 1.4 kcal mol⁻¹, respectively. Of the lower-cost methods studied, M05-2X and MP2 showed the best overall performance with mean absolute deviations of just 1.2 and 1.1 kcal mol⁻¹, respectively. The effect of substitution and position on the acidity of the protons in the various compounds are studied and the structure-reactivity trends in these heterolytic C-H bond dissociation energies (BDEs) are compared with the corresponding homolytic C-H BDEs for the same species.     

Possible Evidence of Amide Bond Formation Between Sinapinic Acid and Lysine-Containing Bacterial Proteins by Matrix-Assisted Laser Desorption/Ionization (MALDI) at 355?nm

We previously reported the apparent formation of matrix adducts of 3,5-dimethoxy-4-hydroxy-cinnamic acid (sinapinic acid or SA) via covalent attachment to disulfide bond-containing proteins (HdeA, Hde, and YbgS) from bacterial cell lysates ionized by matrix-assisted laser desorption/ionization (MALDI) time-of-flight-time-of-flight tandem mass spectrometry (TOF-TOF-MS/MS) and post-source decay (PSD). We also reported the absence of adduct formation when using ??-cyano-4-hydroxycinnamic acid (CHCA) matrix. Further mass spectrometric analysis of disulfide-intact and disulfide-reduced over-expressed HdeA and HdeB proteins from lysates of gene-inserted E. coli plasmids suggests covalent attachment of SA occurs not at cysteine residues but at lysine residues. In this revised hypothesis, the attachment of SA is preceded by formation of a solid phase ammonium carboxylate salt between SA and accessible lysine residues of the protein during sample preparation under acidic conditions. Laser irradiation at 355?nm of the dried sample spot results in equilibrium retrogradation followed by nucleophilic attack by the amine group of lysine at the carbonyl group of SA and subsequent amide bond formation and loss of water. The absence of CHCA adducts suggests that the electron-withdrawing effect of the ??-cyano group of this matrix may inhibit salt formation and/or amide bond formation. This revised hypothesis is supported by dissociative loss of SA (?224?Da) and the amide-bound SA (?206?Da) from SA-adducted HdeA and HdeB ions by MS/MS (PSD). It is proposed that cleavage of the amide-bound SA from the lysine side-chain occurs via rearrangement involving a pentacyclic transition state followed by hydrogen abstraction/migration and loss of 3-(4-hydroxy-3,5-dimethoxyphenyl)prop-2-ynal (?206?Da).     

A universal approach for continuum solvent pK a calculations: are we there yet?

This paper reviews several pK _a calculation strategies that are commonly used in aqueous acidity predictions. Among those investigated were the direct or absolute method, the proton exchange scheme, and the hybrid cluster–continuum (Pliego and Riveros) and implicit–explicit (Kelly, Cramer and Truhlar) models. Additionally, these protocols are applied in the pK _a calculation of 55 neutral organic and inorganic acids in conjunction with various solvent models, including the CPCM-UAKS/UAHF, IPCM, SM6 and COSMO-RS, with a view to identifying a universal approach for accurate pK _a predictions. The results indicate that the direct method is unsuitable for general pK _a calculations, although moderately accurate results (MAD <3 units) are possible for certain classes of acids, depending on the choice of solvent model. The proton exchange scheme generally delivers good results (MAD <2 units), with CPCM-UAKS giving the best performance. Furthermore, the sensitivity of this approach to the choice of reference acid can be substantially lessened if the solvation energies for ionic species are calculated via the IPCM cluster–continuum approach. Reference-independent hybrid approaches that include explicit water molecules can potentially give reasonably accurate values (MAD generally ~2 units) depending on the solvent model and the number of explicit water molecules added.     

Determination of anthelmintic drug residues in milk using ultra high performance liquid chromatography–tandem mass spectrometry with rapid polarity switching

A new UHPLC–MS/MS (ultra high performance liquid chromatography coupled to tandem mass spectrometry) method was developed and validated to detect 38 anthelmintic drug residues, consisting of benzimidazoles, avermectins and flukicides. A modified QuEChERS-type extraction method was developed with an added concentration step to detect most of the analytes at <1 μg kg⁻¹ levels in milk. Anthelmintic residues were extracted into acetonitrile using magnesium sulphate and sodium chloride to induce liquid–liquid partitioning followed by dispersive solid phase extraction for cleanup. The extract was concentrated into dimethyl sulphoxide, which was used as a keeper to ensure analytes remain in solution. Using rapid polarity switching in electrospray ionisation, a single injection was capable of detecting both positively and negatively charged ions in a 13 min run time. The method was validated at two levels: the unapproved use level and at the maximum residue level (MRL) according to Commission Decision (CD) 2002/657/EC criteria. The decision limit (CCα) of the method was in the range of 0.14–1.9 and 11–123 μg kg⁻¹ for drugs validated at unapproved and MRL levels, respectively. The performance of the method was successfully verified for benzimidazoles and levamisole by participating in a proficiency study.