Proton-bound complex mediating retro-Michael-type fragmentation of protonated 3-substituted oxindoles in the Orbitrap high-energy collisional dissociation cell.

RATIONALE: Oxindole derivatives are valuable building blocks for indole chemistry. Systematically exploring the fragmentation behavior of the protonated 3-pyrazole-substituted oxindoles by kinetic methods combined with density functional theory (DFT) calculations is useful for further understanding their basic properties, and might provide some insights into their reactivity trends in synthesis and metabolism. METHODS: All high-resolution high-energy collision-induced dissociation tandem mass spectrometry (CID-MS/MS) experiments were carried out using electrospray ionization hybrid Quadrupole-Orbitrap mass spectrometry in positive ion mode. Theoretical calculations were carried out by the DFT method at the B3LYP level with the 6-311G (d, p) basis set in the Gaussian 03 package of programs. RESULTS: In the fragmentation of protonated 3-pyrazole-substituted oxindoles, the characterized protonated 3-(3-methyl-5-oxo-1H-pyrazol-4(5H)-ylidene)indolin-2-one derivatives and the protonated 5-methylpyrazolone were observed, which were proposed from the cleavage of the C(ß)-C(γ) bond in a retro-Michael reaction. With the kinetic plot, a linear correlation was established between the intensities of this two competitive product ions and the difference in proton affinities of the corresponding neutral molecules, which demonstrated that the retro-Michael reaction was mediated by a proton-bound complex. CONCLUSIONS: Using the kinetic method combined with theoretical calculations, a proton-bound complex mediating retro-Michael reaction was proposed for the fragmentation of protonated 3-pyrazole-substituted oxindoles in the high-energy collisional dissociation tandem mass spectrometry for the first time, which provided potential evidence to further understand their intrinsic bioactivities.

Identifying the proton transfer reaction mechanism via a proton-bound dimeric intermediate for esomeprazoles by a kinetic method combined with density functional theory calculations.

RATIONALE: Esomeprazole analogs are a class of important proton pump inhibitors for the treatment of gastro-esophageal reflux diseases. Understanding the fragmentation reaction mechanism of the protonated esomeprazole analogs will facilitate the characterization of their complex metabolic fate in humans. In this paper, the kinetic method and theoretical calculations were applied to evaluate the fragmentation of protonated esomeprazole analogs. METHODS: All collision-induced dissociation (CID) mass spectrometry experiments were carried out using electrospray ionization (ESI) ion trap mass spectrometry in positive ion mode. Also the accurate masses of fragments were measured on by ESI quadrupole time-of-flight (QTOF) MS in positive ion mode. Theoretical calculations were carried out by the density functional theory (DFT) method with the 6-31G(d) basis set in the Gaussian 03 program. RESULTS: In the fragmentation of the protonated esomeprazole analogs, C-S bond breakage is observed, which gives rise to protonated 2-(sulfinylmethylene)pyridines and protonated benzimidazoles. DFT calculations demonstrate that the nitrogen atom of the pyridine part is the thermodynamically most favorable protonation site, and the C-S bond cleavage is triggered by the transfer of this ionizing proton from the nitrogen atom of the pyridine part to the carbon atom of the benzimidazole part to which the sulfinyl is attached. Moreover, with the kinetic plot, the intensity ratios of two protonated product ions yield a linear relationship with the differences in proton affinities of the corresponding neutral molecules, which provides strong experimental evidence that the reaction proceeds via proton-bound 2-(sulfinylmethylene)pyridine/benzimidazole complex intermediates. CONCLUSIONS: The kinetic method combined with theoretical calculations was successfully applied to probe the proton transfer reaction by proton-bound 2-(sulfinylmethylene)pyridine/benzimidazole complexes in the fragmentation of protonated esomeprazole analogs by ESI CID MS, which is a strong evidence that the kinetic method can be applied in identifying a proton-bound dimeric intermediate in the fragmentation of protonated ions.

Solid-phase extraction based on chloromethylated polystyrene magnetic nanospheres followed by gas chromatography with mass spectrometry to determine phthalate esters in beverages.

An ultrasound-assisted magnetic solid-phase extraction procedure with chloromethylated polystyrene-coated Fe3 O4 nanospheres as magnetic adsorbents has been developed to determine eight phthalate esters (bis(4-methyl-2-pentyl) phthalate, dipentyl phthalate, dihexyl phthalate, benzyl butyl phthalate, bis(2-butoxyethyl) phthalate, dicyclohexyl phthalate, di-n-octyl phthalate, and dinonyl phthalate) simultaneously in beverage samples, in combination with gas chromatography coupled to tandem mass spectrometry for the first time. Several factors related to magnetic solid-phase extraction efficiencies, such as amount of adsorbent, extracting time, ionic strength, and desorption conditions were investigated. The enrichment factors of the method for the eight analytes were over 2482. A good linearity was observed in the range of 10-500 ng/L for bis(2-butoxyethyl) phthalate and 2-500 ng/L for the other phthalate esters with correlation coefficients ranging from 0.9980 to 0.9998. The limits of detection and quantification for the eight phthalate esters were in the range of 0.20-2.90 and 0.67-9.67 ng/L, respectively. The mean recoveries at three spiked levels were 75.8-117.7%, the coefficients of variations were <11.6%. The proposed method was demonstrated to be a simple and efficient technique for the trace analysis of the phthalate esters in beverage samples.

Distinct Bile Acid Profiles in Patients With Chronic Hepatitis B Virus Infection Reveal Metabolic Interplay Between Host, Virus and Gut Microbiome.

Hepatitis B virus (HBV) can hijack the host bile acids (BAs) metabolic pathway during infection in cell and animal models. Additionally, microbiome was known to play critical role in the enterohepatic cycle of BAs. However, the impact of HBV infection and associated gut microbiota on the BA metabolism in chronic hepatitis B (CHB) patients is unknown. This study aimed to unveil the distinct BA profiles in chronic HBV infection (CHB) patients with no or mild hepatic injury, and to explore the relationship between HBV, microbiome and BA metabolism with clinical implications. Methods: Serum BA profiles were compared between CHB patients with normal ALT (CHB-NALT, n = 92), with abnormal ALT (CHB-AALT, n = 34) and healthy controls (HCs, n = 28) using UPLC-MS measurement. Hepatic gene expression in CHB patients were explored using previously published transcriptomic data. Fecal microbiome was compared between 30 CHB-NALT and 30 HCs using 16S rRNA sequencing, and key microbial function was predicted by PICRUSt analysis. Results: Significant higher percentage of conjugated BAs and primary BAs was found in CHB patients even without apparent liver injury. Combinatory BA features can discriminate CHB patients and HCs with high accuracy (AUC = 0.838). Up-regulation of BA importer Na+ taurocholate co-transporting peptide (NTCP) and down-regulation of bile salt export pump (BSEP) was found in CHB-NALT patients. The microbial diversity and abundance of Lactobacillus, Clostridium, Bifidobacterium were lower in CHB-NALT patients compared to healthy controls. Suppressed microbial bile salt hydrolases (BSH), 7-alpha-hydroxysteroid dehydrogenase (hdhA) and 3-dehydro-bile acid Delta 4, 6-reductase (BaiN) activity were found in CHB-NALT patients. Conclusion: This study provides new insight into the BA metabolism influenced both by HBV infection and associated gut microbiome modulations, and may lead to novel strategy for clinical management for chronic HBV infection.

Preclinical pharmacokinetics and tissue distribution of a novel multikinase inhibitor BZG by validated UPLC-MS/MS assay.

A simple and sensitive UPLC-MS/MS assay was developed and validated for rapid determination of BZG in rat plasma and tissues. All biological samples were prepared by protein precipitation method using Imatinib as an internal standard (IS). The analyte and IS were separated on a C18 reverse phase analytical column with 4.5 min of analytical run, at flow rate of 0.3 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) by multiple reactions monitoring (MRM) of the transitions at m/z 451.0→254.0 for BZG and m/z 494.3→394.1 for IS, respectively. The linearity of this method was found to be within the concentration range of 0.5-2500 ng/mL with a lower limit of quantification of 0.5 ng/mL. All validation parameter results were within the acceptable range described in guideline for bioanalytical method validation. The method was successfully applied to a pharmacokinetic and tissue distribution study of BZG in rats. With the preliminary knowledge of in vivo pharmacokinetics and disposition properties, this study will be beneficial for further development of BZG.