Double Derivatization Strategy for High-Sensitivity and High-Coverage Localization of Double Bonds in Free Fatty Acids by Mass Spectrometry.

Free fatty acids (FFAs) are key intermediates of lipid metabolism that have a crucial role in many critical biological processes. The specific locations of carbon-carbon double bonds (C═C) in FFAs are often associated with distinct biological functions. Despite the rapid development of analytical techniques, identification of C═C locations in FFAs with more than three C═C bonds in complex biological matrices remains a challenge. Herein, we describe a double derivatization strategy, coupled with shotgun-mass spectrometry (MS), for unambiguous and sensitive determination of a high-coverage C═C bond (from 1 to 6) locations of FFAs. Our approach is based on combination of acetone labeling of C═C bonds and N,N-diethyl-1,2-ethanediamine (DEEA) labeling of carboxyl groups within FFAs. Acetone labeling of C═C bonds via photochemical reaction provides diagnostic ions, specific to C═C locations, and DEEA labeling of carboxyl groups significantly enhances MS response of diagnostic ions, by invoking a readily protonated tertiary amine group on FFA analytes. By exploiting this double derivatization strategy, the assignment of C═C locations of FFAs with more than three C═C bonds was achieved with high sensitivity (limit of quantitation (LOQ) 0.1-1.5 nmol/L). In contrast, such assignments were not possible by acetone labeling alone, because of the low sensitivity of diagnostic ions in negative ionization mode of MS. The applicability of our method was demonstrated by profiling of FFAs, including unsaturated FFA C═C positional isomers, in liver samples from mice with nonalcoholic fatty liver disease (NAFLD) and their lean controls. The study showed that the high-specificity and high-sensitivity method developed here is promising for accurate identification and quantitation of a wide array of FFAs in biological samples.

Profiling and quantification of aminophospholipids based on chemical derivatization coupled with HPLC-MS.

In this study, a novel strategy based on acetone stable-isotope derivatization coupled with HPLC-MS for profiling and accurate quantification of aminophospholipids (phosphatidylethanolamine and phosphatidylserine) in biological samples was developed. Acetone derivatization leads to alkylation of the primary amino groups of aminophospholipids with an isopropyl moiety; the use of deuterium-labeled acetone (d6-acetone) introduced a 6 Da mass shift that was ideally suited for profiling and quantification analysis with high selectivity and accuracy. After derivatization, significantly increased column efficiency for chromatographic separation and detection sensitivity for MS analysis of aminophospholipids was observed. Furthermore, an accuracy quantification method was developed. Aminophospholipids in biological samples were derivatized with d0-acetone; while more than two aminophospholipid standards were selected for each class of aminophospholipid and derivatized with d6-acetone, which were then used as the internal standards to typically construct a calibration curve for each class to normalize the nonuniformity response caused by the differential fragmentation kinetics resulting from the distinct chemical constitution of individual aminophospholipid species in the biological samples. The excellent applicability of the developed method was validated by profiling and quantification of aminophospholipids presented in liver samples from rats fed with different diets.

Synthesis of magnetic molecularly imprinted polymers for the selective separation and determination of metronidazole in cosmetic samples.

In this study, novel magnetic molecularly imprinted polymers (MMIPs) were developed as a sorbent for solid-phase extraction (SPE) and used for the selective separation of metronidazole (MNZ) in cosmetics; MNZ was detected by high-performance liquid chromatography (HPLC). First, magnetic Fe3O4 nanoparticles (NPs) were prepared by the co-precipitation of Fe(2+)and Fe(3+) ions in an ammonia solution; then oleic acid (OA) was modified onto the surface of Fe3O4NPs. Finally, the MMIP was prepared by aqueous suspension polymerization, involving the copolymerization of Fe3O4NPs@OA with MNZ as the template molecule, methacrylic acid (MAA) as the functional monomer, ethylene glycol maleic rosinate acrylate (EGMRA) as the cross-linking agent, and 2,2-azobisisobutyronitrile (AIBN) as the initiator. The MMIP materials showed high selective adsorption capacity and fast binding kinetics for MNZ; the maximum adsorption amount of the MMIP to MNZ was 46.7 mg/g. The assay showed a linear range from 0.1 to 20.0 µg/mL for MNZ with the correlation coefficient 0.999. The relative standard deviations (RSD) of intra- and inter-day ranging from 0.71 to 2.45% and from 1.06 to 5.20% were obtained. The MMIP can be applied to the enrichment and determination of MNZ in cosmetic products with the recoveries of spiked toner, powder, and cream cosmetic samples ranging from 90.6 to 104.2, 84.1 to 91.4, and 90.3 to 100.4%, respectively, and the RSD was <3.54%.

A comprehensive study of raw and roasted macadamia nuts: Lipid profile, physicochemical, nutritional, and sensory properties.

Macadamia nuts have high nutritional value and positive health attributes. Changes to the composition and availability of these compounds during roasting contribute to product quality. In this study, changes to the chemical composition of lipids (fatty acids, triglycerides, and free fatty acids) and other phytochemicals were analyzed, and a sensory evaluation was carried out of two major varieties of macadamia nuts planted in China, after roasting. Only small changes in fatty acid (FA) content and a slight decrease in total triglycerides (TAGs) were observed after roasting. The free fatty acid (FFA) content and the peroxide value were increased by roasting. The total available polyphenol content increased by 25.6% and the oxidative stability index of kernels increased by 21.6%. The sensory scores for taste and aroma were doubled by roasting. Overall, the sensory, nutritional quality, and oxidative stability of roasted macadamia nuts were greatly improved, compared with raw nuts.

Molecular cloning and characterization of a bifunctional xylanolytic enzyme from Neocallimastix patriciarum.

A cDNA encoding a bifunctional acetylxylan esterase/xylanase, XynS20E, was cloned from the ruminal fungus Neocallimastix patriciarum. A putative conserved domain of carbohydrate esterase family 1 was observed at the N-terminus and a putative conserved domain of glycosyl hydrolase family 11 was detected at the C-terminus of XynS20E. To examine the enzyme activities, XynS20E was expressed in Escherichia coli as a recombinant His(6) fusion protein and purified by immobilized metal ion-affinity chromatography. Response surface modeling combined with central composite design and regression analysis was then applied to determine the optimal temperature and pH conditions of the recombinant XynS20E. The optimal conditions for the highest xylanase activity of the recombinant XynS20E were observed at a temperature of 49 degrees C and a pH of 5.8, while those for the highest carbohydrate esterase activity were observed at a temperature of 58 degrees C and a pH of 8.2. Under the optimal conditions for the enzyme activity, the xylanase and acetylxylan esterase specific activities of the recombinant XynS20E toward birchwood xylan were 128.7 and 873.1 U mg(-1), respectively. To our knowledge, this is the first report of a bifunctional xylanolytic enzyme with acetylxylan esterase and xylanase activities from rumen fungus.