This study examines the scientific basis of a traditional multi-infusion tea-brewing practice common in Asia by evaluating compositional changes, antioxidant capacity, and sensory attributes across successive green tea infusions. Using UPLC-MS, GC-IMS, and elemental profiling, we characterized how repeated extractions alter key tea constituents. Although flavonoids, polyphenols, polysaccharides, and amino acids remained detectable across all infusions, the concentrations of major bioactive compounds including gallic acid, its esters, and L-theanine declined progressively from the first to the third infusion (GT1-GT3), corresponding with reduced in vivo radical-scavenging activity. Changes in volatile profiles and sensory evaluation further indicated noticeable shifts in flavor and taste with each infusion. Pearson correlation analysis revealed that most quantified compounds were positively associated with puckery, bitter, and acidic attributes, and negatively associated with sweetness and freshness. Variable Importance in Projection (VIP) analysis identified L-lysine, L-tyrosine, epicatechin, myricetin, theophylline, quercetin, and epigallocatechin as key contributors to sensory perception. These findings demonstrate that multiple infusions diversify flavor characteristics while modulating the bioactive profile of green tea, offering insights relevant to product development and consumer brewing practices.

It has long been recognised that, in the long term, hazardous acetonitrile (ACN) as a mobile-phase component and the environmentally critical trifluoroacetic acid (TFA) as an ion-pairing reagent should be replaced in high-performance liquid chromatography (HPLC). Nevertheless, methods relying on precisely these reagents are still widely used. Herein, we show that alternative approaches for the analysis of small molecule drugs are readily applicable, using examples from pharmaceutical quality control and medicinal chemistry. First, the principles of green analytical chemistry were implemented in an alternative HPLC method for the theophylline monohydrate monograph of the European Pharmacopoeia, without altering the core methodology. Among the various conditions tested, the use of 2% dimethyl carbonate (DMC) provided equivalent separation and an even slightly improved resolution compared with the original ACN-based pharmacopoeial method. In a second approach, we present a highly sustainable gradient HPLC method for routine purity analysis of synthesis products by replacing TFA with safe, biodegradable, and environmentally benign methanesulfonic acid (MSA). Furthermore, in the case of this gradient method, ACN could be replaced by the biodegradable and environmentally friendly DMC. An eluent composition consisting of 42 parts DMC, 23 parts EtOH, 35 parts H2O and 0.1 parts MSA was found to be equivalent to ACN containing 0.1% TFA. By employing a reproducible protocol using EtOH as a co-solvent, we were able to overcome current challenges associated with the use of organic carbonates in HPLC, which primarily arise from their limited miscibility with water. Given the continued widespread use of gradient HPLC methods employing ACN as an eluent and TFA as an acidic modifier, the methods presented herein offer significant potential to advance the implementation of sustainability in pharmaceutical quality control and medicinal chemistry.

This study aimed to develop strategies to prevent accelerated drug release (alcohol-induced dose dumping) from modified-release matrix tablets in hydroethanolic media. Drugs with different solubility profiles (theophylline, propranolol HCl, paracetamol, and carbamazepine) were formulated with water-soluble or water-insoluble matrix formers, with optional addition of soluble or insoluble fillers. Drug release, medium uptake, and leaching were evaluated in 0.1 N HCl containing 0, 20, or 40% (v/v) ethanol. No release acceleration was observed for theophylline and propranolol HCl, which showed low solubility ratios between hydroethanolic and aqueous media (approximately 2). In contrast, paracetamol and carbamazepine (solubility ratio approximately 20) required formulation adjustments. For paracetamol, comparable release profiles across media were achieved using matrix formers with low medium uptake (e.g., Klucel® MXF or Kollidon® SR) or by incorporating a soluble filler such as lactose. For carbamazepine, the increased solubility in hydroethanolic media shifted the release mechanism from erosion-dominated to diffusion-dominated; similar release profiles were only obtained with hydrophilic polymers exhibiting relatively high erosion rates.

Colorectal cancer (CRC) remains a leading cause of cancer-related mortality worldwide, and improved noninvasive strategies for early detection are urgently needed to complement current screening approaches. We applied an integrated plasma metabolomics workflow that combines untargeted and targeted liquid chromatography-mass spectrometry with machine-learning-assisted feature prioritization. Plasma samples from a discovery cohort of 172 CRC patients and 115 healthy controls were analyzed, followed by validation in an independent cohort of 47 CRC patients and 47 healthy controls. From 146,880 spectral features, random forest analysis was used to robustly rank features, followed by ROC-based prioritization and metabolite annotation. Diagnostic performance was evaluated by using a logistic regression model. Phenotypic assays were conducted to assess the biological activity of selected metabolites in CRC cell models. Five metabolites, N-methylcytisine, 2-piperidone, theophylline, dl-norleucine, and linolenic acid, were consistently reduced in CRC patient plasma across both cohorts. The integrated five-metabolite panel achieved an area under the ROC curve of 0.968 in the validation cohort with a 97.9% sensitivity, an 89.4% specificity, and a 93.7% accuracy. Stratified analyses demonstrated robustness across disease stages and age groups. In vitro assays showed modulation of CRC cell migration and invasion under noncytotoxic conditions. This five-metabolite plasma signature reflects CRC-associated systemic metabolic alterations and demonstrates a strong discriminatory performance. The panel may complement existing screening modalities by contributing to CRC risk stratification and early detection.

Background/Objectives: Population pharmacokinetic (PopPK) modeling in NONMEM requires iterative, expertise-dependent workflows. Naïve zero-shot prompting of general-purpose large language models (LLMs) typically produces NONMEM code that fails to execute. This study introduces PKGPT, a recursive agentic LLM system designed to automate NONMEM-based PopPK model development and benchmarks its performance against human expert models. Methods: PKGPT, powered by Google's Gemini 3.0 Flash, embeds pharmacometrics expertise into phase-specific expert-agent prompts orchestrated across five sequential phases: base model establishment, structural diagnostics, overfitting reduction, random-effects optimization, and covariate analysis. The system recursively executes NONMEM, parses outputs, and iteratively refines control streams. PKGPT was evaluated on three public datasets (warfarin, theophylline, and tobramycin) and benchmarked against independently developed human expert models. Results: PKGPT consistently produced executable, converging NONMEM models across all three datasets. In warfarin, both PKGPT and the human expert selected a one-compartment oral structure (ADVAN2), but the expert achieved a lower OFV (294.41 vs. 484.43) via covariate scaling. In theophylline, PKGPT produced parameter estimates close to the expert solution (Ka = 1.59 vs. 1.46 h-1; CL = 0.0399 vs. 0.0404 L/h/kg). In tobramycin, PKGPT correctly identified a two-compartment structure but produced physiologically implausible peripheral volume estimates (V2 = 149 L vs. expert's 13.2 L). Across datasets, PKGPT did not identify clinically established covariates, and run-to-run reproducibility was variable. Conclusions: PKGPT substantially improves the robustness and usability of LLM-generated NONMEM code compared with naïve zero-shot prompting, accelerating model drafting and iterative refinement, but physiological plausibility and clinical interpretability still require a human-in-the-loop oversight.

Background: Nausea and vomiting in pregnancy affects up to 80% of pregnant women and may progress to hyperemesis gravidarum, leading to maternal morbidity and adverse pregnancy outcomes. Despite numerous pharmacological and non-pharmacological options, the comparative efficacy and safety of these interventions remain unclear. Methods: We conducted a systematic review and network meta-analysis of randomized controlled trials assessing pharmacological and non-pharmacological interventions for nausea and vomiting in pregnancy. The databases searched included CENTRAL, PubMed, and EMBASE (up to 28 May 2024). Eligible trials compared interventions with a placebo in pregnant women with nausea and vomiting in pregnancy. The primary outcomes were symptom severity, assessed using validated tools. Safety outcomes included adverse effects. Data were pooled using frequentist pairwise and network meta-analyses. The risk of bias was assessed using the RoB2 tool, and the certainty of evidence was evaluated using the CINeMA framework. Results: Of 9844 records screened, 24 randomized controlled trials (3017 participants) met the inclusion criteria, encompassing 16 intervention categories. Network analysis ranked quince, vitamin B6 with pomegranate and mint, acupressure P6, dimenhydrinate, and acupuncture combined with doxylamine-pyridoxine as the most effective interventions for reducing symptoms of nausea and vomiting in pregnancy, with considerable uncertainty and low-to-moderate quality of evidence. Reporting of adverse events was limited. Risk of bias was low to moderate. Discussion: Most interventions demonstrated significant benefit over a placebo. However, high heterogeneity and sparse reporting of adverse effects warrant caution when translating these results into clinical practice. Conclusions: This study indicates that both pharmacological (vitamin B6, metoclopramide, dimenhydrinate) and non-pharmacological (ginger, quince, acupressure, acupuncture) interventions might be effective in reducing symptoms of nausea and vomiting in pregnancy.

Critical limits represent quantitative decision thresholds for drugs that require immediate clinician notification and potential life-saving intervention. United States hospitals lack a national standard for drug critical limits. We collected critical limits from 417 US hospitals across all 50 states and Washington, D.C.; of these, 411 maintained drug critical limit lists. We classified hospitals by US Census division and network-affiliated vs. independent status. We applied non-parametric statistical analyses, examined critical limits for 111 drugs, and observed significant inter-institutional variability. Listing frequencies were highest for digoxin 99.8% (410/411), theophylline 72.3% (297/411), lithium 94.9% (390/411), and acetaminophen 86.9% (357/411). Non-therapeutic measurands also appeared, led by ethanol 47.2% (194/411). Kruskal-Wallis analysis revealed highly significant differences (P < 0.01) across census divisions. Mann-Whitney U analysis comparing network vs. independent hospitals yielded significant differences (P < 0.05) in 16 analytes. Qualitative critical values were listed for volatile alcohols and methotrexate. All digoxin critical limits exceeded levels associated with increased hazard ratios. Only 33.9% of hospitals aligned with consensus guidelines for acetaminophen poisoning. Vancomycin and aminoglycoside critical limits showed wide ranges, overlapping peaks and troughs, and random values inconsistently aligned with peak and troughs. Immunosuppressant critical limits often exceeded thresholds associated with toxicity. Psychotropic drugs, including antiepileptics, tricyclic antidepressants, and lithium, demonstrated variability and misalignment relative to consensus guidelines. Drugs of abuse did not appear in critical notification lists. Results provide hospitals with references that will enhance hospital notification practices and patient safety. We encourage sharing of critical notification lists to foster future research efforts and enhance standards of care.

Although mechanochemical synthesis offers a sustainable way to produce solid forms of pharmaceutical compounds, the molecular-level mechanisms that govern solvent-mediated transformations remain largely unexplored. In this study, we present an in situ solid-state nuclear magnetic resonance (NMR) approach to directly monitor the evolution of liquid-assisted grinding reactions under magic-angle spinning conditions. Using a modified CLASSIC NMR protocol, we tracked the cocrystallization of two model systems, theophylline-benzamide and metronidazole-gallic acid, in the presence of solvents with different levels of polarity. This method allows us to observe both the solid and liquid phases within the reaction environment simultaneously, revealing transient intermediates, hydrate formation, and solvent-dependent polymorphic outcomes. Comparisons with time-resolved in situ X-ray diffraction confirm the complementary nature of NMR in capturing mechanistic details that are otherwise inaccessible to a diffraction-based analysis. This work establishes solid-state NMR as a powerful and accessible in situ tool for investigating the effects of solvents in mechanochemical synthesis, thereby advancing our molecular understanding of polymorphic control and reaction pathways.

Caffeine, theophylline and theobromine are methylxanthines, a class of alkaloids present in coffee, tea and cocoa and therefore widely consumed all around the world. They exhibit several biological properties making them useful ingredients in cosmetics, as food additives or in pharmaceutical formulations. However, their solubility in water is limited. Extraction and purification often require the use of petrochemical volatile organic solvents. In the present paper, the solubility in water of caffeine, theophylline and theobromine in the presence of various natural solubilizing agents is reported. Niacinamide, as well as sodium or cholinium levulinate, salicylate, ferulate or caffeate were used to enhance the solubility of these methylxanthines in water. For example, solubility of theobromine was found to increase by a factor of 300 in aqueous solutions of ferulate salt. Solubilization mechanisms were investigated experimentally using 1H NMR and 2D ROESY-NMR and theoretically using molecular dynamics simulations. Both studies point towards the presence of π-stacking-type interactions between methylxanthines and solubilizing compounds. Benzene-based moieties exhibit stronger interactions and π-stacking with caffeine than pyridine-based moieties, such as nicotinic acid and niacinamide. These compounds can therefore be considered as complexing agents for methylxanthines rather than hydrotropes. Importantly, the linear dependence of solubility on solubilizing agent concentration indicates that these complexes maintain their solubilized state upon dilution, thereby enabling complete miscibility in water without precipitation.

Coffee influences multiple physiological processes, including gut function, stress, cognition, and the microbiome. However, the mechanisms underlying these effects remain poorly understood. In this study, we examined coffee's impact on the microbiota-gut-brain axis-a bidirectional communication pathway between the gut microbiome and the brain-and assessed whether these effects occur independently of caffeine in healthy participants. Our primary outcome was microbiota composition and function, whereas the secondary outcome was gut microbial metabolites and coffee-related metabolites (NCT05927038 and NCT05927103). Significant group differences emerged in faecal microbiome composition, with coffee drinkers showing increased relative abundance of Cryptobacterium and Eggerthella species, alongside reduced levels of the metabolite's indole-3-propionic acid, indole-3-carboxyaldehyde, and the neurotransmitter γ-aminobutyric acid. Behaviourally, coffee drinkers exhibited greater impulsivity and emotional reactivity, whereas non-coffee drinkers demonstrated better memory performance. Some alterations in the faecal metabolome were reversible following coffee abstinence, and reintroduction triggered acute microbiome changes independent of caffeine. An integrated model identified nine key metabolites-including theophylline, caffeine, and selected phenolic acids-strongly linked to microbial species and cognitive measures. These findings reveal previously unrecognised effects of coffee on the microbiota-gut-brain axis, suggesting that microbiome profiles could potentially predict coffee consumption patterns and highlighting a close association between coffee intake and gut microbial composition.

Caffeine (CAF) is one of the most widely consumed psychoactive substances worldwide. It is primarily metabolized to paraxanthine (PAR), theobromine (THR), and theophylline (THY). While CAF metabolism has been extensively characterized in humans and rodent models, corresponding data in zebrafish (Danio rerio) larvae remain limited. This study provides a comprehensive characterization of CAF biotransformation in zebrafish larvae using validated LC-QQQ-MS for quantitative metabolite profiling and FTIR imaging for spatially resolved tissue distribution mapping. Zebrafish larvae at 4 days post fertilization (dpf) were exposed to CAF at concentrations of 15, 25, and 50 mg/L for 18 h. The LC-MS/MS analysis demonstrated dose-dependent conversion of CAF to PAR (up to 4.54%), THR (up to 1.32%), and THY (up to 0.27%). The PAR/CAF and THR/CAF ratios increased as CAF concentration rose, while the THY/CAF ratio declined. In parallel, FTIR imaging was applied to visualize the spatial distribution of CAF and selected metabolites in larval tissue sections, confirming the presence of CAF, PAR, and THR and revealing their localization within the tissue. However, THY was not detected by this method. Metabolite localization differed across larval tissues, indicating compartmentalized metabolic processes. This study provides the first comprehensive characterization of CAF metabolism in zebrafish larvae, revealing low but detectable CYP450-mediated metabolic activity prior to full hepatic maturation. The findings support the utility of zebrafish larvae as a model for developmental pharmacokinetic studies.

A rapid method utilizing matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been developed and validated for the quantification of methylxanthines in beverages. Beverage samples were directly analyzed by MS after dilution, with the entire procedure completed within two minutes. Caffeine-D3 and 1-methylxanthine were used as internal standards for the quantitative analysis of caffeine, theophylline, and theobromine, which exhibited strong linear correlations (R[2] ≥ 0.9978). By integrating the concentration ratios of the isomeric compounds theophylline and theobromine derived from two diagnostic ions generated through MALDI tandem mass spectrometry, this approach enables the absolute quantification of both analytes. The method demonstrated satisfactory accuracy, with values from the spiked beverage samples ranging from 84.7% to 119.5%. Furthermore, real beverage samples were analyzed using both the developed method and the conventional high-performance liquid chromatography (HPLC) approach, yielding comparable results. The proposed method provides a rapid, accurate, and high-throughput analysis of methylxanthines in beverages.

Low-dose theophylline has anti-inflammatory effects, but its efficacy in mild to moderate COPD remains controversial.