Free D-amino acids produced by commensal bacteria in the colonic lumen.

D-amino acids (D-AAs) have various biological activities, such as activation of N-methyl-D-aspartic acid (NMDA) receptor as a co-agonist by D-Ser. Since several free D-AAs are released in the broth monocultured with bacterium and D-AAs are probably utilized for bacterial communication, we presume that intestinal microbiota releases several kinds of free D-AAs, which may be involved in the hosts' health. However, presently, only four free D-AAs have been found in the ceacal lumen, but not in the colonic lumen. Here, we showed, by simultaneous analysis of chiral AAs using high-sensitivity liquid chromatography-tandem mass spectrometry (LC-MS/MS), that 12 free D-AAs (D-Ala, D-Arg, D-Asp, D-Gln, D-Glu, D-allo-Ile, D-Leu, D-Lys, D-Met, D-Phe, D-Ser, and D-Trp) are produced by intestinal microbiota and identified bacterial groups belonging to Firmicutes as the relevant bacterial candidates.

Adsorption of Pluronic Surfactants in Alkylene Carbonates on Silica.

Adsorption of triblock Pluronic surfactants bearing poly(ethylene oxide) (PEO) chains of different lengths, L-62 (5 EO groups on each end), L-64 (13 EO groups on each end), and F-68 (79 EO groups on each end), on silica has been characterized using atomic force microscopy (AFM). The solvent used herein was a mixture of ethylene carbonate (EC) and propylene carbonate (PC). The three Pluronic surfactants were dissolved in the mixed solvent, with the PEO chain acting as a solvophilic group and the poly(propylene oxide) (PPO) chain acting as a solvophobic group. The approaching force curve measurements for the three Pluronics (at 10 mmol dm-3) revealed repulsive forces from an apparent separation of 20-30 nm. The most solvophilic Pluronic surfactant with the longest PEO chain (F-68) showed continuously increasing repulsive interaction with decreasing separation. The Milner-Witten-Cates (MWC) theory described the repulsive force curve data of F-68, suggesting that F-68 forms a polymer brush on the silica surface. The retracting force curve measurements detected stretching forces for the three Pluronic systems. These stretching forces were observed more frequently for the L-62 system than for the F-68 system, but the pull-off distance was shorter for L-62 than for F-68.

Autophagy promotes degradation of internalized collagen and regulates distribution of focal adhesions to suppress cell adhesion.

Adhesion of cells to the extracellular matrix (ECM) via focal adhesions (FAs) is crucial for cell survival, migration, and differentiation. Although the regulation of FAs, including by integrins and the ECM, is important to cell behavior, how FAs are regulated is not well known. Autophagy is induced by both cell adhesion and cell detachment. Here, we showed that autophagosomes are located close to internalized collagen and paxillin, which is a well-known marker of FAs. Autophagy-deficient cells showed increased levels of internalized collagen compared with control cells. Moreover, paxillin exhibited a more peripheral distribution and the area of paxillin was increased, and adhesion-induced focal adhesion kinase signaling was impaired and adhesion was enhanced, in autophagy-deficient cells. These results suggest that autophagy suppressed cell adhesion by regulating internalized ECM and FAs.

Automatic on-line solid-phase extraction with ultra-high performance liquid chromatography and tandem mass spectrometry for the determination of ten antipsychotics in human plasma.

An automatic on-line solid-phase extraction with ultra-high performance liquid chromatography and tandem mass spectrometry method was developed for the simultaneous determination of ten antipsychotics in human plasma. The plasma sample after filtration was injected directly into the system without any pretreatment. A Shim-pack MAYI-C8 (G) column was used as a solid-phase extraction column, and all the analytes were separated on a Shim-pack XR-ODS III column with a mobile phase consisting of 0.1% v/v formic acid in water with 5 mM ammonium acetate and acetonitrile. The method features were systematically investigated, including extraction conditions, desorption conditions, the equilibration solution, the valve switching time, and the dilution for column-head stacking. Under the optimized conditions, the whole analysis procedure took only 10 min. The limits of quantitation were in the range of 0.00321-2.75 µg/L and the recoveries ranged from 75.9 to 122%. Compared with the off-line ultra-high performance liquid chromatography and the reported methods, this validated on-line method showed significant advantages such as minimal pretreatment, shortest analysis time, and highest sensitivity. The results indicated that this automatic on-line method was rapid, sensitive, and reliable for the determination of antipsychotics in plasma and could be extended to other target analytes in biological samples.

Pre-column dilution large volume injection ultra-high performance liquid chromatography-tandem mass spectrometry for the analysis of multi-class pesticides in cabbages.

Pre-column dilution large volume injection (PD-LVI), a novel sample injection technique for reverse phase ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), was developed in this study. The PD-LVI UHPLC-MS/MS system was designed by slightly modifying the commercial UHPLC-MS/MS equipment with a mixer chamber. During the procedure of PD-LVI, sample solution of 200µL was directly carried by the organic mobile phase to the mixer and diluted with the aqueous mobile phase. After the mixture was introduced to the UHPLC column in a mobile phase of acetonitrile-water (15/85, v/v), the target analytes were stacked on the head of the column until following separation. Using QuEChERS extraction, no additional steps such as solvent evaporation or residue redissolution were needed before injection. The features of PD-LVI UHPLC-MS/MS system were systematically investigated, including the injection volume, the mixer volume, the precondition time and the gradient elution. The efficiency of this approach was demonstrated by direct analysis of 24 pesticides in cabbages. Under the optimized conditions, low limits of detection (0.00074-0.8 ng/kg) were obtained. The recoveries were in the range of 63.3-109% with relative standard deviations less than 8.1%. Compared with common UHPLC-MS/MS technique, PD-LVI UHPLC-MS/MS showed significant advantages such as excellent sensitivity and reliability. The mechanism of PD-LVI was demonstrated to be based on the column-head stacking effect with pre-column dilution. Based on the results, PD-LVI as a simple and effective sample injection technique of reverse phase UHPLC-MS/MS for the analysis of trace analytes in complex samples showed a great promising prospect.

Quantitative Analysis of Chloramphenicol in Royal Jelly by Column-switching LC-MS/MS Using a Pretreatment Column with a Higher-pressure Capability.

An on-line pretreatment liquid chromatography-tandem mass spectrometry (LC-MS/MS) system was developed for the analysis of chloramphenicol (CAP) in royal jelly. A novel methylcellulose-immobilized restricted access media column with a higher-pressure capability of 60 MPa (MC-ODS HP) was developed for the effective removal of proteins and other compounds in the sample matrix. CAP in a sample solution was extracted in 2 min by the column-switching LC-MS/MS system. The system provides a minimum sample pretreatment along with highly sensitive and reproducible analysis. As a result, the limit of quantitation of CAP was 10 pg/mL (= 0.1 µg/kg royal jelly) and the linear dynamic range was between 10 and 10000 pg/mL (correlation coefficient greater than 0.999). The proposed method meets the requirements of regulations in EU (0.3 µg/kg). The inter-day precision and accuracy of CAP at 100 pg/mL over 3 days were 4.5 and 95.4%, respectively. Compared with the conventional method with a pressure of below 25 MPa, the peak separation in the MRM chromatogram was improved by using smaller particles (1.6 µm) for the analytical ODS column. The LC-MS/MS system with an MC-ODS HP expanded the applicability of the automated pretreatment.

An automatic versatile system integrating solid-phase extraction with ultra-high performance liquid chromatography-tandem mass spectrometry using a dual-dilution strategy for direct analysis of auxins in plant extracts.

An automatic versatile system which integrated solid phase extraction (SPE) with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed. Diverse commercial SPE columns can be used under an ambient pressure in this online system realized by a dual-dilution strategy. The first dilution enabled the direct injection of complex samples with minimal pretreatment, and the second dilution realized direct introduction of large volume of strong eluent into the UHPLC column without causing peak broadening or distortion. In addition, a post-column compensation mode was also designed for the matrix-effects evaluation. The features of the online system were systematically investigated, including the dilution effect, the capture of desorption solution, the column-head stacking effect and the system recovery. Compared with the offline UHPLC system, this online system showed significant advantages such as larger injection volume, higher sensitivity, shorter analysis time and better repeatability. The feasibility of the system was demonstrated by the direct analysis of three auxins from different plant tissues, including leaves of Dracaena sanderiana, buds and petals of Bauhinia. Under the optimized conditions, the whole analysis procedure took only 7min. All the correlation coefficients were greater than 0.9987, the limits of detection and the limits of quantitation were in the range of 0.560-0.800ng/g and 1.80-2.60ng/g, respectively. The recoveries of the real samples ranged from 61.0 to 117%. Finally, the post-column compensation mode was applied and no matrix-effects were observed under the analysis conditions. The automatic versatile system was rapid, sensitive and reliable. We expect this system could be extended to other target analytes in complex samples utilizing diverse SPE columns.

Analysis of impurities in streptomycin and dihydrostreptomycin by hydrophilic interaction chromatography/electrospray ionization quadrupole ion trap/time-of-flight mass spectrometry.

Impurities in streptomycin (STR) and dihydrostreptomycin (DHS) were investigated by hydrophilic interaction chromatography/electrospray ionization quadrupole ion trap/time-of-flight mass spectrometry (HILIC/ESI-QIT/TOFMS). Samples were separated on a fused-core silica column (100 mmx2.1 mm i.d., particle size: 2.7 microm) with isocratic elution using 200 mM ammonium formate buffer (pH 4.5) and acetonitrile as mobile phase. Constant neutral loss survey in accurate mass measurement was carried out by QIT/TOFMS. Formulae, chemical structures of impurities in an STR sample were suggested with supporting results on the probable pathways of STR biosynthesis by Streptomyces griseus.

On-line pretreatment using methylcellulose-immobilized cation-exchange restricted access media for direct liquid chromatography/mass spectrometric determination of basic drugs in plasma.

A novel methylcellulose-immobilized restricted access media column with strong cation-exchange groups on an internal surface (MC-SCX) was evaluated for the direct injection analysis of basic polar drugs in plasma by column-switching liquid chromatography/mass spectrometry (LC/MS). Analytical conditions, including an automated pretreatment step and MS detection, were optimized for a series of basic drugs (doxepin, desipramine, imipramine, nortriptyline, amitriptyline, clomipramine). On-line pretreatment with the MC-SCX column followed by fast gradient analysis using a C18 column resulted in a total analysis cycle time of 7 min for each spiked plasma sample. More than 150 plasma samples spiked with target compounds were measured without compromising MS detection (relative standard deviations less than 11% for all compounds, and regression coefficients greater than 0.99).

Direct analysis of drugs in plasma by column-switching liquid chromatography-mass spectrometry using a methylcellulose-immobilized reversed-phase pretreatment column.

A novel methylcellulose-immobilized reversed-phase pretreatment column (MC-ODS) for column switching liquid chromatography-mass spectrometry (LC-MS) was investigated to improve recovery and durability. Pretreatment and analytical conditions were optimized so that high throughput and high selectivity was ensured during mass spectrometric analysis. Analytical runs, including deproteinization and gradient LC analysis, were conducted in a 6-min cycle. As a consequence, recoveries for test drugs (metoprolol, propranolol, lidocaine, dibucaine, bupivacaine) were greater than 90% and more than 300 plasma samples spiked with target compounds were directly injected and measured without compromising MS detection or system performance.