Deep eutectic solvents (DESs) have garnered significant attention in pharmaceutical research in recent years due to their high efficiency, facile separability and biocompatibility. Characterised by hydrogen-bond donors (HBDs) and hydrogen-bond acceptors (HBAs), DESs exhibit low toxicity, favourable biodegradability, and excellent solubilising properties, rendering them promising alternatives to conventional organic solvents in drug synthesis, natural active ingredient extraction and drug delivery. This review systematically evaluates the broad pharmaceutical applications of DESs, with particular emphasis on enhanced drug solubility and bioavailability. In plant extraction, DESs outperform traditional solvents (e.g., diethyl ether) by improving the recovery efficiency of bioactive compounds. In drug synthesis, certain DESs function simultaneously as reaction media and catalysts, enhancing reaction efficiency and selectivity while minimising environmental impact. In drug delivery, DESs facilitate transdermal and oral absorption through interactions with biological membranes, making improved delivery efficiency. Despite these advantages, challenges remain, including high viscosity, formulation complexity, and unresolved regulatory considerations. Future research must focus on physico-chemical optimisation, safety evaluation, and scalable production to fully realise the potential in pharmaceutical applications.
Electron paramagnetic resonance (EPR) spectroscopy has long been established across various scientific disciplines for characterizing organic radicals, organometallic complexes, protein structures and dynamics, polymerization processes, and radical degradation phenomena. Despite its extensive utility in these areas, EPR spectroscopy's application within pharmaceutical science has historically been constrained, primarily due to factors such as high equipment costs, a steep learning curve, complex spectral deconvolution and analysis, and a traditional lack of emphasis on single-electron chemistry in pharmaceutical research. This review aims to provide a thorough examination of EPR spectroscopy's applications in analyzing a wide array of para-magnetic species relevant to pharmaceutical research. We detail how EPR spectroscopy can be employed to assess free radical scavenging properties in pharmaceutical compounds, elucidate drug mechanisms of action, and explore pharmacokinetics. Additionally, we investigate the role of free radicals in drug-induced toxicity and drug-membrane interactions, while also covering the application of EPR spectroscopy in drug delivery research, advanced studies of metallodrugs, and monitoring of oxygen levels in biological systems through EPR oximetry. The recent advancements in the miniaturization of EPR spectro meters have paved the way for their application in on-site and in-line mo nitoring during the manufacturing process and quality control of pharmaceutical substances and final drug formulations due to being the only direct and non-invasive detection technique for radical detection. Through these discussions, we highlight the substantial contributions of EPR spectroscopy to the advancement of pharmaceutical sciences.
Converting macrocycle lactams into bicyclic lactams is proposed as an additional way to further increase the metabolic stability of peptide-based drugs. Unfortunately, the synthesis of bicyclic lactams has to start almost from scratch. This study explores the Hofmann-Löffler-Freytag (HLF) reaction mechanism and products as a potential late-stage functionalisation strategy for facile conversion of macrocyclic to bicyclic ring. Laurolactam, a macrocyclic amide, exhibits significant potential for transformation into bioactive bicyclic structures with smaller, β-, γ-, δ-, and ε-lactam rings, further increasing rigidity and hydrolytic stability. With irradiation provided by a 370 nm lamp, light-induced rearrangement reaction was monitored using nuclear magnetic resonance (NMR), while involved radical intermediates were trapped using N-tert-butyl-α-phenylnitrone (PBN) spin-trap and characterised via EPR. While only two radical adduct types were identified in the electron para magnetic resonance (EPR) (C-centered radical and chlorine radical), all eight possible products are observed in the NMR. Quantum chemical calculations provide deeper insights into reaction thermodynamics and kinetics, explaining why the N-centered radical was not observed. This research highlights the feasibility of using the HLF reaction to transform macrocyclic lactams into stable bicyclic drug candidates, paving the way for new therapeutic developments.
This study aimed to examine the motility, biofilm production, endotoxin release, and antibiotic resistance of 81 Ralstonia pickettii isolates collected from different pharmaceutical water systems in Croatia. Swimming and twitching motility were detected in all isolates, while swarming was not observed. Biofilm production was detected in approximately 40 % of the isolates under the tested conditions. Notably, extracellular polymeric substance (EPS) production was a common trait among all isolates. Endotoxin production was detected with the Limulus Amoebocyte Lysate test. Antibiotic susceptibility testing revealed consistent resistance to colistin, as well as significant resistance rates to β-lactam antibiotics, ertapenem, amoxicillin/clavulanic acid, ticarcillin and ampicillin. High susceptibility to first-generation cephalosporins, cephalexin, cefoxitin and chloramphenicol was observed. All isolates were susceptible to tigecycline and tetracycline. The isolates were grouped into three genetically closely related clusters, yet notable phenotypic diversity in biofilm production and antibiotic susceptibility persisted within these groups. The study highlights R. pickettii's adaptability in pharmaceutical water systems, marked by its motility, biofilm-forming capabilities, and multidrug resistance. These results emphasise the importance of rigorous monitoring of water systems to reduce transmission risks and prevent the emergence of resistant strains in clinical environments.
Daratumumab is a fully human anti-CD38 monoclonal antibody with strong potential as a targeting vector for therapeutic radionuclides. This study aimed to develop a freeze-dried daratumumab immunoconjugate kit by selecting a suitable chelator (DOTA-NHS, p-SCN-Bn-DOTA, or p-SCN-Bn-1B4M-DTPA) for the 177Lu-labelling, optimising the freeze-drying formulation, and evaluating the physicochemical properties and purity profiles. Conjugation performed in carbonate buffer at elevated temperature enhanced chelator incorporation and supported the selection of daratumumab-p-SCN-Bn-DOTA as the most suitable candidate, achieving radiolabelling yield up to 99.8 % without additional purification. Among the evaluated freeze-dried formulations, a saline-based, buffer-free sucrose-mannitol formulation containing polysorbate 20 (S.F5) provided the most favourable characteristics, including minimal residual moisture and the highest monomer purity with non-detectable HMWS species under the applied SE-HPLC conditions. ATR-FTIR and Raman spectroscopy confirmed preservation of the antibody structural integrity after conjugation and freeze-drying. In an in vitro study using human serum, [177Lu]Lu-daratumumab-p-SCNBn-DOTA maintained higher radiochemical purity for 168 h than [177Lu]Lu-daratumumab-p-SCN-Bn-1B4MDTPA, indicating greater stability. These results support the feasibility of a ready-to-use freeze-dried daratumumab-p-SCN-Bn-DOTA kit for the 177Lu-labelling.
This study investigates the development and characterisation of niosome-based delivery systems for olanzapine, an antipsychotic drug. Niosomes were prepared using various grades of Span surfactants (Span 60, Span 40, and Span 20) in combination with cholesterol at different ratios. The formulations were characterised in terms of particle size, polydispersity index, zeta potential, and encapsulation efficiency. Results showed an inverse relationship between surfactant hydrophilic-lipophilic balance (HLB) values and niosome size, with Span 60 producing the smallest vesicles. Optimal formulations were achieved with a 1:1 ratio of surfactant to cholesterol. Span 60 niosomes exhibited the highest encapsulation efficiency (up to 81 ± 2.5 %) and the most negative zeta potential, indicating superior stability. In vitro release studies demonstrated sustained release profiles for all niosomal formulations compared to the free drug, with Span 60 formulations showing the slowest release rates. Release kinetics analysis revealed a Fickian diffusion-controlled mechanism best described by the Korsmeyer-Peppas model. These findings suggest that niosomal formulations, particularly those based on Span 60, offer a promising approach for improving olanzapine delivery, potentially enhancing its bioavailability and therapeutic efficacy in the treatment of psychiatric disorders.
The objective of this study was to explore the protective effect of astragaloside IV on a model of isoproterenol-induced (ISO) hyper-trophic injury in rat cardiomyocytes H9c2 (cell line derived from embryonic BD1X rat heart tissue). A cell hypertrophy injury model was established (H9c2 cells treated with 100 μmol L-1 ISO). The cells were divided into normal control, a model group, and an astragalo-side IV group at several concentrations. Astragaloside IV was pre-administered for 2 hours, followed by ISO treatment for 24 hours. Cell viability, cell surface area, apoptosis rate, lactate dehydrogenase (LDH) activity, reactive oxygen species (ROS), superoxide dismutase (SOD), the mRNA levels of Bcl-2, Bax, p62, and LC3, the protein expressions of Sirt1, p62, caspase-3, beclin, and p53 and the LC3II/LC3I ratio were detected. Astragaloside IV significantly alleviated ISO-induced hypertrophy injury in H9c2 cells, reduced cell surface area and LDH release, decreased apoptosis rate and intracellular ROS levels, increased SOD levels, upregulated the expressions of autophagy-related mRNA and proteins, and downregulated the expressions of apoptosis-related mRNA and proteins. Astragaloside IV can effectively inhibit ISO-induced hypertrophy and apoptosis in H9c2 cells, and its mechanism may be related to promoting auto-phagy and reducing oxidative stress.
During in-use stability testing of cefixime granules for oral suspension, an impurity with a relative retention time of 0.19 was consistently detected and increased during storage. To ensure regulatory compliance and patient safety, the impurity was structurally identified and toxicologically qualified. A laboratory-scale formulation and commercial products were studied under refrigerated and ambient conditions. The impurity was isolated by automated fraction collection and characterised by liquid chromatography-mass spectrometry and tandem mass spectrometry. Kinetic evaluation showed pseudo-first-order formation, with faster accumulation at ambient temperature. The impurity was identified as a γ-lactone degradation product of cefixime, present as multiple stereoisomers stabilised under acidic conditions. In silico toxicological assessment using complementary platforms indicated no additional structural alerts, no mutagenic potential, and negligible acute toxicity. The impurity forms only after prolonged storage of reconstituted suspensions and is classified as an ICH M7 Class 5 impurity, requiring no further genotoxicity testing. The applied analytical-computational workflow provides an efficient approach for impurity qualification in β-lactam antibiotics.
Sedation is crucial for managing mechanically ventilated intensive care unit (ICU) patients, but agents differ in their effects. Propofol, benzodiazepines and α 2-agonists are commonly used, yet their comparative impact remains unclear. This review searched OVID MEDLINE and Embase from January 2006 to June 2025 for randomised controlled trials in adult ICU patients. The primary outcome was duration of mechanical ventilation; secondary outcomes were ICU length of stay, delirium and mortality. Twenty-six trials (N = 4,993) were included. Dexmedetomidine significantly shortened mechanical ventilation (mean difference [MD] -0.60 days; 95 % CI -0.89 to -0.31), with larger effects versus midazolam (MD -1.25 days) and mixed comparators (MD -1.23 days), but not versus propofol (MD -0.34 days). ICU stay was also reduced (MD -0.94 days; 95 % CI -1.49 to -0.39). Delirium risk decreased (odds ratio [OR] 0.58; 95 % CI 0.38-0.87). No mortality difference was found. Dexmedetomidine is therefore associated with a modest but clinically meaningful reduction in ventilation time, ICU stay and delirium, particularly when compared with benzodiaze-pines, though benefits over propofol are less certain.
Umbilical cord mesenchymal stem cells (UC-MSCs) have shown therapeutic potential in renal diseases due to their homing ability. This study compared the effects of three administration routes (intravenous, local renal injection, and interventional injection) on UC-MSC distribution in kidney tissue. Eighteen New Zealand rabbits were assigned to the three groups (n = 6 each), and DiI-labelled UC-MSCs were tracked using confocal microscopy to evaluate their distribution in the kidney, lung, and brain. Local renal injection led to high MSC concentrations at the injection site, but distribution to the contralateral kidney was minimal and comparable to that of intravenous injection. Intravenous delivery via the marginal ear vein was simple and convenient but resulted in limited renal homing (< 1 %) and no significant difference between kidneys. Interventional injection achieved the highest delivery efficiency (12.4 %) and a more uniform renal distribution. Notably, inflammatory cytokine levels (IL-6, TNF-α, IL-10) were significantly elevated in the local injection group (p < 0.05). These results indicated that the choice of administration route critically affects MSC targeting and therapeutic potential, and interventional injection may offer the most effective strategy for precise UC-MSC delivery in renal therapy.
Alzheimer's disease (AD) is characterised by β-amyloid (Aβ) plaque accumulation and tau hyperphosphorylation. H3 relaxin, a neuro-peptide, is known to exert neuroprotective effects. In this study, we investigated how H3 relaxin confers neuroprotection in a streptozotocin (STZ)-induced mouse model and modulates PI3K/Akt-mTOR signalling. Mice were divided into four groups (n = 6 per group): control (saline), STZ, STZ + H3 relaxin, and STZ + donepezil. Following STZ induction, H3 relaxin (1 µg per day) was administered intracerebro ventricularly (ICV) for 14 consecutive days, whereas donepezil (2.5 mg kg-1 per day) was administered orally for the same duration. Cognitive performance was assessed using the Morris water maze (MWM) test. Aβ deposition in the cortex was evaluated through immunohistochemistry. Western blotting was conducted for tau phospho rylation, PI3K/Akt/mTOR signalling, and autophagy markers in the hippocampus. Oxidative stress and inflammation markers were measured using ELISA. H3 relaxin markedly improved memory by decreasing escape latency and duration while spending more time in the target quadrant in the MWM test. Additionally, H3 relaxin reduced Aβ plaque burden and tau phosphorylation (Ser396/404) while enhancing PI3K/Akt-mTOR signalling. Oxidative stress was attenuated, as evidenced by increased GSH and HO-1 levels and reduced MDA and H2O2 concentrations. Moreover, markers of inflammation, NF-κB and TNF-α were suppressed. Overall, H3 relaxin ameliorated cognitive deficits in STZ-induced AD mice through modulation of impaired PI3K/Akt-mTOR signalling, reduction of Aβ and tau pathology, and promotion of autophagy.
This study examined the effects of omega-3 fatty acids supplementation on gut barrier integrity, systemic inflammation, neurotrans-mission and oxidative stress, in an aged rat model of propionic acid (PPA)-induced neurotoxicity. Twenty-four aged male rats were divided into four groups: control, omega-3, PPA and PPA + omega-3. Serum cytokines, tight-junction proteins (TJP1), dopamine, serotonin, short-chain fatty acids (SCFAs), oxidative stress markers, and histopathology of the brain and small intestine were evaluated. PPA exposure significantly increased tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and reduced TJP1 expression, confirming gut barrier disruption and systemic inflammation. Omega-3 fatty acids supplementation selectively reduced IL-6 but did not reverse PPA-induced TNF-α elevation or oxidative stress. CLDN2 expression increased in PPA + omega-3 rats, suggesting a compensatory but incomplete barrier response. Dopamine, serotonin, and SCFA levels showed upward trends with supplementation but were not statistically significant. Histological analysis demonstrated partial preservation of neuronal and intestinal structure in the PPA + omega-3 group. Overall, omega-3 fatty acids exerted modest anti-inflammatory effects but failed to fully restore oxidative balance or barrier integrity in aged rats, suggesting that omega-3 fatty acids may be more effective as a preventive rather than restorative intervention in ageing-related gut-brain axis disruption.
This study assessed the gastrointestinal (GI) safety profiles of tigecycline, omadacycline, and eravacycline through a retrospective disproportionality analysis of reports submitted to the FDA Adverse Event Reporting System (FAERS) between the second quarter of 2005 and the first quarter of 2024. Among 3,261 adverse event reports associated with these agents, 809 (24.8 %) involved gastrointestinal disorders, with tigecycline accounting for the largest proportion (588 reports), followed by omadacycline (197) and eravacycline (24). Disproportionality analysis revealed that gastrointestinal disorders ranked among the top three system organ classes for all three drugs, with positive signals observed for tigecycline (ROR = 1.63), omadacycline (ROR = 3.04), and eravacycline (ROR = 1.79), the strongest association being with omadacycline. While most GI events were consistent with known safety information, several unexpected signals were identified, including gastrointestinal haemorrhage, melena, small-intestinal obstruction, tongue discolouration, and intestinal perforation for tigecycline, as well as lip swelling and tongue discolouration for omadacycline. The median onset times of GI events were 4, 0, and 2.5 days for tigecycline, omadacycline, and eravacycline, respectively, with nearly half of the events occurring within three days of treatment initiation. These findings reveal distinct GI safety patterns among newer tetracycline-derived antibiotics and underscore the importance of early and route-specific monitoring in clinical practice.
Cell division cycle 20 homologue (Cdc20), a key regulator of the anaphase-promoting complex/cyclosome (APC/C), is frequently overexpressed in human cancers and represents a promising therapeutic target. However, monotherapy targeting Cdc20 has shown limited efficacy, partly due to compensatory activation of cyclin-dependent kinase 1 (Cdk1). In this study, we investigated the combinatorial potential of the pan-Cdk inhibitor ZK304709 with the Cdc20 inhibitor apcin in HeLa cervical cancer cells. Transcriptomic analysis revealed that both CDC20 and CDK1 are upregulated in cervical cancer tissues. Mechanistically, apcin treatment induced cyclin B1 accumulation and enhanced Cdk1 phosphorylation at Thr161, suggesting feedback activation. In contrast, ZK304709 reduced p-Cdk1(T161) levels and suppressed Cdc20 expression at both protein and mRNA levels. Functionally, the combination of apcin and ZK304709 synergistically inhibited cell proliferation and induced G2/M phase arrest in HeLa cells. These findings demonstrate that dual inhibition of Cdk1 and Cdc20 disrupts compensatory signalling pathways and enhances antitumour efficacy in HeLa cells, providing a rational strategy for combination therapy in cervical cancer.
The effects of vilazodone (VZD) on the acquisition of active avoidance behavior were examined in C57BL/6J mice. Both female and male mice were assigned to three groups (n = 8 per group per sex): the vehicle control group (VEH), the 0.5 mg kg-1 vilazodone lower dose group (VZD0.5) and the 1 mg kg-1 vilazodone higher dose group (VZD1.0). Spontaneous locomotion and anxiety-like behaviour were assessed after drug administration intraperitoneally in an open field test (OFT). Another cohort of mice was trained in a three-day shuttle box active avoidance test (AAT) after drug administrations with the aim of evaluating the effects of VZD on the acquisition of active avoidance behaviour. In the OFT, VZD decreased freezing time in the corner area in both female and male mice, indicating reduced anxiety-like behaviours. In the AAT, the active avoidance rate was significantly improved on day 1 in female mice and day 2 in male mice, suggesting that VZD facilitated active avoidance learning with sexual dimorphism. Furthermore, the increased active avoidance rates were negatively correlated with freezing time during training. Interestingly, these group differences and correlations diminished on day 3, implying that the facilitation was restricted to early training phases. Collectively, VZD facilitates the acquisition of active avoidance behaviour in mice with distinct sexual dimorphism and temporal dynamics.
Glucocorticoids are a group of drugs increasingly used in modern medical practice due to their pronounced anti--inflammatory and immunosuppressive properties. In this study, prednisolone disodium phosphate and prednisolone acetate were analysed, with the aim of developing and validating an HPLC method in accordance with ICH Q2(R2) guidelines and quantifying their content in the active pharmaceutical ingredient powder and a model in--house sample. By applying the HPLC-DAD method with gradient elution, effective separation of the analytes was achieved. The method met all validation parameter requirements. The obtained results showed that the content of both analytes in the tested samples (bulk API powders and in-house prepared model formulation) was within the prescribed limits according to current pharmacopoeial standards. The proposed HPLC-DAD method was assessed for its applicability and environmental profile utilizing a range of green and blue metric tools. This comprehensive evaluation confirms that the method adheres to green analytical principles, making it suitable for sustainable pharmaceutical analysis.
Methyl caffeate (MC), a naturally occurring methyl ester of caffeic acid (CA), exhibits potent antioxidant activity and a broad spectrum of biological effects. This study investigates the antioxidant mechanism of MC through its reaction with the stable radical DPPH•, employing both experimental and computational approaches. Kinetic measurements were conducted in a predo minantly nonpolar medium (1,4-dioxane with phosphate buffer), revealing concerted proton and electron transfer. This experimental evidence was supported by values of kinetic isotope effects (KIEs) and thermodynamic activation parameters. Analysis of the intrinsic bond orbitals (IBOs) along the calculated intrinsic reaction coordinate (IRC) trajectories supported the proposed proton- coupled electron transfer (PCET) reaction mechanism. Additionally, the Fe(II) complexation ability of MC was evaluated spectrophotometrically, demonstrating stable complex formation at pH 7.0, suggesting potential for mitigating hydroxyl radical generation in physiological conditions. These findings offer new insights into the antioxidant behaviour of MC and its potential applications in pharmaceutical and nutraceutical formulations.
This study evaluates the antihyperlipidemic (pancreatic lipase inhibition assay), antiglycation (inhibition of bovine serum albumin glycation, BSA glycation), and antioxidant activity (ABTS, DPPH and FRAP assays) of ethanolic extracts from flowering parts of five widely distributed plant species in Croatia - Crocus heuffelianus Herb. (tepals), Nicotiana tabacum L. (petals), Malva sylvestris L. (petals), Calendula officinalis L. and Helianthus annuus L. (both sterile ligulate flowers). An in vitro-simulated system of human digestion was employed to assess the bioaccessibility of the selected phenolics and the stability of the extracts' antioxidant, hypolipi demic, and antiglycation potential following each digestion phase. The concentrations of l-ascorbic acid, individual flavonoids, and phenolic acids were determined using RP-HPLC analysis. Principal component analysis revealed significant differences in the content of bioactive compounds and their biological activity among the investigated plant species. All original extracts exhibited high antioxidant capacity (> 70 %) in at least one assay, with N. tabacum and H. annuus demonstrating the strongest anti-oxidant capacity throughout digestion. H. annuus contained the highest levels of total identified phenolic acids, total identified phenols, and total identified compounds, while N. tabacum and C. heuffelianus exhibited the highest total flavonoid content. Among individual compounds, protocatechuic acid, quercetin, and ferulic acid significantly contributed to antioxidant activity. N. tabacum had the strongest antihyperlipidemic potential in the original extracts, as well as in the most digestion phases. Strong BSA glycation inhibition (70-100 %) was observed in all plant extracts across various digestion phases, with the exception of C. heuffelianus, which exhibited mode rate inhibitory effects. These findings suggest that the analyzed flower-derived plant materials, some of which are often considered agricultural waste, can serve as sustainable and valuable resources of bioactive compounds for functional food, dietary supplements, and pharmaceutical applications.
Diabetic retinopathy (DR) is one of the primary causes of vision impairment, affecting individuals with diabetes, and is marked by the neurodegeneration of the retina along with increased intraocular pressure (IOP). This study sought to determine the effects of pelargonidin on extracellular matrix (ECM) modulation and the inhibition of transforming growth factor-β (TGF-β) and Janus Kinase 2/Signal Transducer and Activator of Transcription 3 (JAK2/STAT3) pathway in retinal ganglion cells of streptozotocin-induced diabetic rats. Male Sprague-Dawley rats (180-200 g) were rendered diabetic by intraperitoneal administration of streptozotocin (STZ). The rats were divided into 5 groups: control, diabetic model (STZ), STZ + low dose pelargonidin (12.5 mg kg-1 per day), STZ + medium dose pelargonidin (25 mg kg-1 per day) and STZ + high dose pelargonidin (50 mg kg-1 per day). IOP was monitored using a tonometer. Whole-mount retinal immunofluorescence staining using RNA-binding protein with multiple splicing (RBPMS) was performed to assess retinal ganglion cell (RGC) density. Protein expression levels of apoptotic markers, ECM components, and TGF-β and JAK2/STAT3 signalling pathways were evaluated by Western blotting. Pelargonidin treatment dose-dependently reduced the elevated IOP. Importantly, immunofluorescence analysis revealed a marked dose-dependent preservation of retinal ganglion cell (RGC) density: STZ-induced RGC loss was significantly reversed by pelargonidin, with the highest dose restoring RGC density to near-control or higher levels in both the central and peripheral retina. This was achieved via modulation of apoptosis-related proteins through the upregulation of Bcl-xL, Bcl-2, and downregulation of Bad, Bax and cleaved caspase-3. Furthermore, pelargonidin modulated ECM remodelling protein expression in the RGC layer. In particular, TGF-β2/Smad2/3 signalling was downregulated, and the JAK2/STAT3 pathway was upregulated. By reducing IOP, preserving RGC density, modulating ECM deposition, inhibiting TGF-β and upregulating the JAK2/STAT3 pathway, pelargonidin exerts protective effects against diabetic retinal injury. The results of this study further confirm the pharmacological potential of pelargonidin as a therapeutic agent for diabetic retinopathy.
The quality of chamomile (Matricaria recutita) is largely determined by its content of essential oils and flavonoids, the main pharmacologically active constituents. In this study, the phyto-chemical profiling of 22 commercially available chamomile flower tea samples was aided by chemometrics, comparing loose teas of whole heads with tea bags containing comminuted flowers. Principal component analysis (PCA) and agglomerative hierarchical clustering (AHC), which included both essential oil and flavonoid constituents, showed that chamomile teas can be well-differentiated and categorised into two groups that are closely related to the pharmaceutical form and largely explain the influence of processing. Multivariate analyses of the phytochemical data matrix showed clear differences between loose chamomile tea and tea bags, with the former having a more consistent composition and an overall higher quality. The essential oil content varied widely (0.75-5.34 mL kg-1), with only five loose teas exceeding the minimum content specified in the European Pharmacopoeia (≥ 4 mL kg-1), while most tea bag samples did not fulfil this requirement. GC-MS analyses of essential oils revealed sesquiterpenes as predominant constituents, assigning all samples to the bisabolol oxide-rich chemotype. The total flavonoid content determined by UV/Vis spectrophotometry ranged from 0.17 to 0.55 %, whereas RP-HPLC/DAD analysis revealed that the levels of apigenin-7-glucoside in tea bag samples often did not meet pharmacopoeial standards. Partial least squares-discriminant analysis (PLS-DA) yielded a robust and statistically significant model, showing for the first time that the quality differences between loose teas and tea bags can be explained by at least four key components. These results highlight the utility of chemometric tools in chamomile quality assessment and emphasise the need for improved standardisation that supports the preference for whole flower teas to ensure therapeutic efficacy.