Efficient Drug Loading Method for Poorly Water-Soluble Drug into Bicelles through Passive Diffusion.

The bicelle, a type of solid lipid nanoparticle, comprises phospholipids with varying alkyl chain lengths and possesses the ability to solubilize poorly water-soluble drugs. Bicelle preparation is complicated and time-consuming because conventional drug-loading methods in bicelles require multiple rounds of thermal cycling or co-grinding with drugs and lipids. In this study, we proposed a simple drug-loading method for bicelles that utilizes passive diffusion. Drug-unloaded bicelles were placed inside a dialysis device and incubated in a saturated solution of ketoconazole (KTZ), which is a model drug. KTZ was successfully loaded into bare bicelles over time with morphological changes, and the final encapsulated concentration was dependent on the lipid concentration of the bicelles. When polyethylene glycol (PEG) chains of two different lengths (PEG2K and 5K) were incorporated into bicelles, PEG2k and PEG5k bicelles mitigated the morphological changes and improved the encapsulation rate. This mitigation of morphological changes enhanced the encapsulated drug concentration. Specifically, PEG5k bicelles, which exhibited the greatest prevention of morphological changes, had a lower encapsulated concentration after 24 h than that of PEG2k bicelles, indicating that PEGylation with a longer PEG chain length improved the loading capacity but decreased the encapsulation rate owing to the presence of a hydration layer of PEG. Thus, PEG with a certain length is more suitable for passive loading. Moreover, loading factors, such as temperature and vehicles used in the encapsulation process, affected the encapsulation rate of the drug. Taken together, the passive loading method offers high throughput with minimal resources, making it a potentially valuable approach during early drug development phases.

A Review on the Foodomics Based on Liquid Chromatography Mass Spectrometry.

Due to the globalization of food production and distribution, the food chain has become increasingly complex, making it more difficult to evaluate unexpected food changes. Therefore, establishing sensitive, robust, and cost-effective analytical platforms to efficiently extract and analyze the food-chemicals in complex food matrices is essential, however, challenging. LC/MS-based metabolomics is the key to obtain a broad overview of human metabolism and understand novel food science. Various metabolomics approaches (e.g., targeted and/or untargeted) and sample preparation techniques in food analysis have their own advantages and limitations. Selecting an analytical platform that matches the characteristics of the analytes is important for food analysis. This review highlighted the recent trends and applications of metabolomics based on "foodomics" by LC-MS and provides the perspectives and insights into the methodology and various sample preparation techniques in food analysis.

Experimental design of a stable isotope labeling derivatized UHPLC-MS/MS method for the detection/quantification of primary/secondary bile acids in biofluids.

An efficient analytical platform is required to characterize the human metabolome in pathology. For this purpose, ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) combined with chemical derivatization stands out as one of the most powerful techniques. A targeted metabolomics platform for 11 bile acids (BAs) profiling in human serum and bile samples using a stable isotope labeling derivatization (SILD) was applied. For SILD, the design of experiments (DoE) was employed to optimize the reaction conditions such five factors in three levels. The sample preparation built upon a liquid-liquid extraction requiring small volumes (20 µL). In application, the relation between the BA and short-chain fatty acid levels in human serum and bile samples from patients with bile duct diseases were investigated. The proposed method offers significant utility in the large-scale biological analyses of hepato-biliary-pancreatic-related diseases.

LC-MS/MS assay for the investigation of acetylated Alpha-synuclein in serum from postmortem Alzheimer's disease pathology.

Alpha-synuclein (α-Syn), a neuronal protein, has been linked to the inflammation and development of neurodegenerative diseases. In a number of neurodegenerations, α-Syn has been investigated in the central nervous system and cerebrospinal fluid. However, there are few studies concerning the variations in peripheral α-Syn in postmortem Alzheimer's disease (AD) pathology. In this study, the quantitative procedure for the determination of peripheral acetylated α-Syn regarding N-terminal amino acid's site (α-Syn1-6; MDVFMK and Ac-α-Syn1-6; Ac-MDVFMK) was developed using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and tryptic digestion without antibody. Serum samples were selected from postmortem specimens based on autopsy pathological examination of AD remark. The LC-MS/MS assay with ACQUITY UPLC BEH C18 column was applied on the basis of electrospray positive ionization. When subjected to N-terminal α-Syn peptides using MonoSpin Typsin HP preparation, doubly- and singly-charged α-Syn1-6 and Ac-α-Syn1-6 ions were observed at m/z 386 > 104 and m/z 813 > 72, respectively, which correspond to quantitative profiling with internal standards. In the calibration, the range of 10-1000 nmol/L showed r2 = 0.999 and recovery from 86.0% to 115.0% (RSD < 9.0%). Using this procedure, peripheral α-Syn1-6 from serum samples could not be detected. On the other hand, Ac-α-Syn1-6 levels were measured from 106.9 to 319.8 nmol/L (AD; n = 10) and 147.1-292.0 nmol/L (control; n = 10) with an insignificant difference. From these preliminary results, individual Ac-α-Syn levels in serum were inferred with nonspecific biomarker regarding to AD pathology.

Stable Isotope Labeling by Carbon-13 in Bacteria Culture for the Analysis of Residual Avermectin Using Stable Isotope Dilution Liquid Chromatography Tandem Mass Spectrometry.

This study describes the development of a new stable isotope labeling method by carbon-13 in bacteria culture (SILCB) for the analysis of residual antibiotics via liquid chromatography with tandem mass spectrometry (LC-MS/MS). Stable isotope dilution (SID) LC-MS/MS with QuEChERS was employed to determine avermectin, particularly avermectin B1a (AV-a) and B1b (AV-b), based on completely 13C-labeled internal standards (13C-ISs) obtained from the SILCB. Our SILCB was developed from an optimal inorganic medium using 13C6-glucose for Streptomyces avermitilis (14893 strain). A rough extract containing 13C-ISs was purified via high-speed countercurrent chromatography with a volatile two-phase solvent system composed of n-hexane/ethyl acetate/methanol/0.5% formic acid in water (7/3/5/5/, V/V). The purified 13C-ISs were evaluated to confirm the presence of completely 13C-labeled ions with m/z 938 > 326 and m/z 923 > 309 for AV-a and AV-b, respectively. The QuEChERS approach with the 13C-ISs procedure achieved acceptable recovery rates in beef meat samples of 99.5 - 100.0% (RSD < 2.0%, n = 6). For the analysis of residual antibiotics in foodstuffs by SID-LC-MS/MS and QuEChERS, the SILCB represents a significant improvement over previous methods suffering from cumbersome sample preparation and matrix effects.

Theanine, Antistress Amino Acid in Tea Leaves, Causes Hippocampal Metabolic Changes and Antidepressant Effects in Stress-Loaded Mice.

By comprehensively measuring changes in metabolites in the hippocampus of stress-loaded mice, we investigated the reasons for stress vulnerability and the effect of theanine, i.e., an abundant amino acid in tea leaves, on the metabolism. Stress sensitivity was higher in senescence-accelerated mouse prone 10 (SAMP10) mice than in normal ddY mice when these mice were loaded with stress on the basis of territorial consciousness in males. Group housing was used as the low-stress condition reference. Among the statistically altered metabolites, depression-related kynurenine and excitability-related histamine were significantly higher in SAMP10 mice than in ddY mice. In contrast, carnosine, which has antidepressant-like activity, and ornithine, which has antistress effects, were significantly lower in SAMP10 mice than in ddY mice. The ingestion of theanine, an excellent antistress amino acid, modulated the levels of kynurenine, histamine, and carnosine only in the stress-loaded SAMP10 mice and not in the group-housing mice. Depression-like behavior was suppressed in mice that had ingested theanine only under stress loading. Taken together, changes in these metabolites, such as kynurenine, histamine, carnosine, and ornithine, were suggested to be associated with the stress vulnerability and depression-like behavior of stressed SAMP10 mice. It was also shown that theanine action appears in the metabolism of mice only under stress loading.

Theanine, the Main Amino Acid in Tea, Prevents Stress-Induced Brain Atrophy by Modifying Early Stress Responses.

Chronic stress can impair the health of human brains. An important strategy that may prevent the accumulation of stress may be the consumption of functional foods. When senescence-accelerated mice prone 10 (SAMP10), a stress-sensitive strain, were loaded with stress using imposed male mouse territoriality, brain volume decreased. However, in mice that ingested theanine (6 mg/kg), the main amino acid in tea leaves, brain atrophy was suppressed, even under stress. On the other hand, brain atrophy was not clearly observed in a mouse strain that aged normally (Slc:ddY). The expression level of the transcription factor Npas4 (neuronal PAS domain protein 4), which regulates the formation and maintenance of inhibitory synapses in response to excitatory synaptic activity, decreased in the hippocampus and prefrontal cortex of stressed SAMP10 mice, but increased in mice that ingested theanine. Lipocalin 2 (Lcn2), the expression of which increased in response to stress, was significantly high in the hippocampus and prefrontal cortex of stressed SAMP10 mice, but not in mice that ingested theanine. These data suggest that Npas4 and Lcn2 are involved in the brain atrophy and stress vulnerability of SAMP10 mice, which are prevented by the consumption of theanine, causing changes in the expression of these genes.

Comprehensive quantification of purine and pyrimidine metabolism in Alzheimer's disease postmortem cerebrospinal fluid by LC-MS/MS with metal-free column.

The metabolome presence of nucleobases, nucleosides, nucleotides and related phosphorylated metabolites has been examined for Alzheimer's disease (AD). Although reversed-phase liquid chromatography tandem mass spectrometry (LC-MS/MS) has been used for the determination of these analytes, they were limited in chromatographic signal intensity and reproducibility owing to significant peak tailing caused by complexing with metallic cations and phosphate groups. In this work, we applied LC-MS/MS analysis with a metal-free column for comprehensive quantification of 40 analytes regarding to purine and pyrimidine metabolism in postmortem cerebrospinal fluid (pCSF) from AD patients. For the analytical column, an InertSustain AQ-C18 metal-free PEEK column was used. MS detection was by electrospray positive ionization. The metal-free column allowed for sharp peak detection of highly polar metabolites within a running time of 17 min. In validation, the limits of detection (LOD), the limit of quantitation (LOQ) and recovery value using a pooled pCSF sample are 1-500 nM, 0.5-250 nM and a range of 53.1-144.0% (RSD ranged from 0.4 to 19.6%). The developed LC-MS/MS method utilizing a metal-free column provides an accurate quantification of some metabolites regarding purine and pyrimidine metabolism in pCSF samples obtained from AD patients.

Widely targeted metabolomics of Alzheimer's disease postmortem cerebrospinal fluid based on 9-fluorenylmethyl chloroformate derivatized ultra-high performance liquid chromatography tandem mass spectrometry.

Confirmed biomarkers of postmortem cerebrospinal fluid (pCSF) are used to differentiate between Alzheimer's disease (AD) patients and healthy seniors with high diagnostic accuracy. However, the extent to which the performance of specific metabolic profiling facilitates reliable estimations of the concentrations of the different pCSF biomarkers and their ratios remains unclear. The interpretation of the lower levels of molecules of metabolic profiling and their concentration ratios in pCSF related to brain disorders could facilitate an unchallenging detection of peripheral biomarkers of AD stages and other dementia types. In this study, we proposed the use of widely targeted metabolomics for pCSF metabolic profiling using 9-fluorenylmethyl chloroformate- (FMOC) derivatized ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to evaluate the diversity of 97 amine-mediated metabolic patterns and pathways from confirmed diagnosis based on AD brain pathology. Our results identified the metabolites that contributed toward and mutually influenced the principal component analysis plot with integrated analytes. Furthermore, the AD group showed a significant variation in several analyte concentration levels compared to those of control subjects. These trends of the concentration levels expressed by the amine metabolic pathways indicated the decreased activity of polyamine and tryptophan-kynurenine (Trp-Kyn) metabolisms. Moreover, increased metabolites such as methionine sulfoxide, 3-methoxy-anthranilate, cadaverine, guanine, and histamine were observed by widely targeted metabolomics of pCSF from the AD subjects. According to their metabolic pathway analysis using FMOC-derivatized UHPLC-MS/MS assay, we supposed that the involvement of polyamine and Trp-Kyn metabolisms was observed in the pCSF samples.