Single ion mobility monitoring (SIM2) stitching method for high-throughput and high ion mobility resolution chiral analysis.

Chiral analysis is of high interest in many fields such as chemistry, pharmaceuticals and metabolomics. Mass spectrometry and ion mobility spectrometry are useful analytical tools, although they cannot be used as stand-alone methods. Here, we propose an efficient strategy for the enantiomer characterization of amino acids (AAs) using non-covalent copper complexes. A single ion mobility monitoring (SIM2) method was applied on a TIMS-ToF mass spectrometer to maximize the detection and mobility separation of isomers. Almost all of the 19 pairs of proteinogenic AA enantiomers could be separated with at least one combination with the chiral references L-Phe and L-Pro. Furthermore, we extended the targeted SIM2 method by stitching several mobility ranges, in order to be able to analyze complex mixtures in a single acquisition while maintaining high mobility resolution. Most of the enantiomeric pairs of AAs separated with the SIM2 method were also detected with this approach. The SIM2 stitching method thus opens the way to a more comprehensive chiral analysis with TIMS-ToF instruments.

Enantioselective Reduction of Noncovalent Complexes of Amino Acids with CuII via Resonant Collision-Induced Dissociation: Collision Energy, Activation Duration Effects, and RRKM Modeling.

Formation of noncovalent complexes is one of the approaches to perform chiral analysis with mass spectrometry. Enantiomeric distinction of amino acids (AAs) based on the relative rate constants of competitive fragmentations of quaternary copper complexes is an efficient method for chiral differentiation. Here, we studied the complex [CuII,(Phe,PhG,Pro-H)]+ (m/z 493) under resonant collision-induced dissociation conditions while varying the activation time. The precursor ion can yield two main fragments through the loss of the non-natural AA phenylglycine (PhG): the expected product ion [CuII,(Phe,Pro-H)]+ (m/z 342) and the reduced product ion [CuI,(Phe,Pro)]+ (m/z 343). Enantioselective reduction describes the difference in relative abundance of these ions, which depends on the chirality of the precursor ion: the formation of the reduced ion m/z 343 is favored in homochiral complexes (DDD) compared to heterochiral complexes (such as LDD). Energy-resolved mass spectrometry data show that reduction, which arises from rearrangement, is favored at a low collision energy (CE) and long activation time (ActT), whereas direct cleavage preferentially occurs at a high CE and short ActT. These results were confirmed with kinetic modeling based on RRKM theory. For this modeling, it was necessary to set a pre-exponential factor as a reference, so that the E0 values obtained are relative values. Interestingly, these simulations showed that the critical energy E0 required to form the reduced ion is comparable in both homochiral and heterochiral complexes. However, the formation of product ion m/z 342 through direct cleavage is associated with a lower E0 in heterochiral complexes. Consequently, enantioselectivity would not be caused by enhanced reduction in homochiral complexes but rather by direct cleavage being favored in heterochiral complexes.

Novel natural and synthetic inhibitors of solute carriers SGLT1 and SGLT2.

Selective analogs of the natural glycoside phloridzin are marketed drugs that reduce hyperglycemia in diabetes by inhibiting the active sodium glucose cotransporter SGLT2 in the kidneys. In addition, intestinal SGLT1 is now recognized as a target for glycemic control. To expand available type 2 diabetes remedies, we aimed to find novel SGLT1 inhibitors beyond the chemical space of glycosides. We screened a bioactive compound library for SGLT1 inhibitors and tested primary hits and additional structurally similar molecules on SGLT1 and SGLT2 (SGLT1/2). Novel SGLT1/2 inhibitors were discovered in separate chemical clusters of natural and synthetic compounds. These have IC50-values in the 10-100 µmol/L range. The most potent identified novel inhibitors from different chemical clusters are (SGLT1-IC50 Mean ± SD, SGLT2-IC50 Mean ± SD): (+)-pteryxin (12 ± 2 µmol/L, 9 ± 4 µmol/L), (+)-ε-viniferin (58 ± 18 µmol/L, 110 µmol/L), quinidine (62 µmol/L, 56 µmol/L), cloperastine (9 ± 3 µmol/L, 9 ± 7 µmol/L), bepridil (10 ± 5 µmol/L, 14 ± 12 µmol/L), trihexyphenidyl (12 ± 1 µmol/L, 20 ± 13 µmol/L) and bupivacaine (23 ± 14 µmol/L, 43 ± 29 µmol/L). The discovered natural inhibitors may be further investigated as new potential (prophylactic) agents for controlling dietary glucose uptake. The new diverse structure activity data can provide a starting point for the optimization of novel SGLT1/2 inhibitors and support the development of virtual SGLT1/2 inhibitor screening models.

Intestinal phase-II metabolism of quercetin in HT29 cells, 3D human intestinal tissues and in healthy volunteers: a qualitative comparison using LC-IMS-MS and LC-HRMS.

Flavonoids are a large class of dietary molecules, among which quercetin is the most ubiquitous, which undergo an extensive intestinal phase-II metabolism. We compared the in vivo metabolism of quercetin in healthy volunteers with two in vitro models, HT29 cells and 3 D human intestinal tissues. Supernatants of the in vitro experiments and the human intestinal fluids (HIF) were analyzed by LC-IMS-MS and LC-HRMS in a qualitative way. Quercetin glucuronides, sulfates and their methyl conjugates were detected in all three systems. The metabolic profiles were found to be different, both in terms of the metabolites produced and their relative proportions. In particular, quercetin sulfates were almost absent in supernatants from HT29 cells incubations while they were a major metabolite in HIF and also found in 3 D intestinal tissues incubations. IMS provided structural information as well as a third dimension of characterization, while HRMS brought increased sensitivity and MS/MS confirmation. HT29 cells are a useful tool to generate phase-II metabolites but do not represent the in vivo situation. 3 D intestinal tissues appear as a more relevant tool to study the intestinal phase-II metabolism of flavonoids.

Intestinal disposition of quercetin and its phase-II metabolites after oral administration in healthy volunteers.

OBJECTIVES: Quercetin is one of the main dietary flavonoids and undergoes a substantial intestinal phase-II metabolism. Quercetin conjugates have been detected in plasma and in urine, but their presence in the small intestine has not been assessed. This study aimed to investigate the intestinal metabolism and metabolite excretion of quercetin by the human small intestinal wall after oral dosing. METHODS: Six healthy volunteers were given a capsule of 500 mg of quercetin with 240 ml of water. Duodenal fluids were collected using the intraluminal sampling technique for 4 h and analysed by LC-MS/MS. KEY FINDINGS: Phase-II metabolites of quercetin were detected and quantified in aspirated intestinal fluids. Metabolites appeared almost immediately after administration, indicating an intestinal metabolism and apical excretion into the lumen. Quercetin-3'-O-glucuronide was found to be the main intestinal metabolite. Our results could not conclude on the enterohepatic recycling of quercetin or its metabolites, although several individual profiles showed distinctive peaks. CONCLUSIONS: This study highlights the intestinal metabolism and excretion of quercetin and its conjugates in humans and gives insights into the relevant concentrations which should be used to investigate potential food-drug interactions in vitro.

Identification of phase-II metabolites of flavonoids by liquid chromatography-ion-mobility spectrometry-mass spectrometry.

Flavonoids are a class of natural compounds with a broad range of potentially beneficial health properties. They are subjected to an extensive intestinal phase-II metabolism, i.e., conjugation to glucuronic acid, sulfate, and methyl groups. Flavonoids and their metabolites can interact with drug transporters and thus interfere with drug absorption, causing food-drug interactions. The site of metabolism plays a key role in the activity, but the identification of the various metabolites remains a challenge. Here, we developed an analytical method to identify the phase-II metabolites of structurally similar flavonoids. We used liquid chromatography-ion-mobility spectrometry-mass spectrometry (LC-IMS-MS) analysis to identify phase-II metabolites of flavonols, flavones, and catechins produced by HT29 cells. We showed that IMS could bring valuable structural information on the different positional isomers of the flavonols and flavones. The position of the glucuronide moiety had a strong influence on the collision cross section (CCS) of the metabolites, with only minor contribution of hydroxyl and methyl moieties. For the catechins, fragmentation data obtained from MS/MS analysis appeared more useful than IMS to determine the structure of the metabolites, mostly due to the high number of metabolites formed. Nevertheless, CCS information as a molecular fingerprint proved to be useful to identify peaks from complex mixtures. LC-IMS-MS thus appears as a valuable tool for the identification of phase-II metabolites of flavonoids. Graphical abstract Structural identification of phase-II metabolites of flavonoids using LC-IMS-MS.