Separation, identification, and fingerprinting of antioxidant components in persimmon (Diospyros kaki) leaves by offline two-dimensional liquid chromatography with electrochemical detection and tandem mass spectrometry.

In this work, the antioxidant components in persimmon (Diospyros kaki) leaves were separated by offline two-dimensional liquid chromatography-electrochemical detection (LC×LC-ECD) and identified by LC-tandem mass spectrometry (LC-MS/MS). A total of 33 antioxidants, mainly proanthocyanidins, and glycosides of kaempferol and quercetin, were identified. The antioxidant assays demonstrated that the fractions collected from the first-dimension LC (1D-LC) possessed considerable radical scavenging capabilities, with correlation coefficients of peak area versus radical scavenging capability of 1,1-diphenyl-2-picrylhydrazyl and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) being 0.9335 and 0.9116, respectively. The fingerprinting showed that 37 peaks were present in all samples. The major antioxidant components of persimmon leaves were the glycosides of kaempferol and quercetin. Finally, fourteen antioxidants were quantitatively assessed. Offline LC×LC provided high peak capacity and separation; ECD enabled specific screening and detection of antioxidant components; and MS/MS provided excellent identification capability. In this study, the combination of the three approaches was utilized to screen for antioxidant components in persimmon leaves, with satisfactory findings. In conclusion, this technique is an effective means for rapid analysis of antioxidant components and quality control of medicinal plants, achieving rapid separation of congeners and facilitating more accurate qualitative and quantitative analyses.

High-Mass Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Absolute Quantitation of Noncovalent Protein-Protein Binding Interactions.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a robust and powerful tool for studying biomacromolecules and their interactions. However, quantitative detection of high-mass analytes (kDa to MDa range) remains challenging for MALDI-MS. Herein, we successfully used commercially available purified proteins (ß-galactosidase and BSA) as internal standards for high-mass MALDI-MS analysis and achieved absolute quantification of several high-mass analytes. We systematically evaluated four sample deposition methods, and using the sandwich deposition method with saturated sinapinic acid as the top layer, we performed a robust quantitative analysis by high-mass MALDI-MS. Combined with chemical cross-linking, this quantitative strategy was further used to evaluate the affinity of protein-protein interactions (PPIs), specifically of two soluble protein receptors (interleukin 1 receptor and interleukin 2 receptor) and two membrane protein receptors (rhodopsin and angiotensin 2 receptor 1) with their interaction partners. The measured dissociation constants of the protein complexes formed were between 10 nM and 5 µM. We expect this high-throughput, rapid method, which does not require labeling or immobilization of any of the interaction partners, to become a viable alternative to traditional biophysical methods for studying PPIs.

Closing the gap: Ion chromatography coupled to high-resolution mass spectrometry to trace highly polar anionic substances in groundwater.

Persistent, mobile and toxic (PMT), and very persistent and very mobile (vPvM) substances pose a threat to the water cycle but are often not covered in conventional environmental monitoring programs. Within this realm of substances, one compound class of concern are pesticides and their transformation products as they are deliberately introduced into the environment. To detect very polar anionic substances, including many pesticide transformation products with log DOW values ranging between -7.4 and 2.2, an ion chromatography high-resolution mass spectrometry method was developed in this study. Since inorganic anions, such as chloride and sulfate, interfere with the analysis of organic species, their removal via precipitation with Ba/Ag/H cartridges was assessed. To improve LOQs, vacuum-assisted evaporative concentration (VEC) was evaluated. By using VEC and removing inorganic salt ions, the median LOQ improved from 100 ng/L in evian® water without sample treatment to 10 ng/L after enrichment and 30 ng/L in karst groundwater. Using this method, twelve out of 64 substances covered by the final method were found in karst groundwater in concentrations of up to 5600 ng/L, and seven exceeded 100 ng/L. To the authors' knowledge, the dimethenamid TP M31 and chlorothalonil TP SYN548008 were detected for the first time in groundwater samples. The coupling to a high-resolution mass spectrometer also allows for non-target screening and hence, this method presents a powerful tool to tackle PMT/vPvM substances.