PEG-Lipids: Quantitative Study of Unimers and Aggregates Thereof by the Methods of Molecular Hydrodynamics.

Understanding the polymorphism of lipids in solution is the key to the development of intracellular delivery systems. Here, we study the dynamics of poly(ethylene glycol)-lipid (PEG-Lipid) conjugates aiming at a better understanding of their molecular properties and aggregation behavior in solution. Those PEG-Lipids are used as components of lipid nanoparticles (LNPs). LNPs are gaining increased popularity, e.g., by their utilization in modern vaccination strategies against SARS-CoV-2. Characterization of the systems is conducted by the classical methods of hydrodynamics in different solvents, such as ethanol and water, which are also commonly used for LNP formulation. We were able to elucidate the structurally associated hydrodynamic properties of isolated PEG-Lipids in ethanol, revealing the typically expected values of the hydrodynamic invariant for random coil polymers. By virtue of the same experimental setting, the PEG-Lipids' behavior in water was as well studied, which is a less good solvent than ethanol for the PEG-Lipids. Our experiments demonstrate that PEG-Lipids dissolved in water form well-defined micelles that can quantitatively be characterized in terms of their degree of aggregation of PEG-Lipid polymer unimers, their hydrodynamic size, and solvation, i.e., the quantitative determination of water contained or associated to the identified micelles. Quantitative results obtained from classical hydrodynamic analyses are fully supported by studies with standard dynamic light scattering (DLS). The obtained diffusion coefficients and hydrodynamic sizes are in excellent agreement with numerical results derived from analytical ultracentrifugation (AUC) data. Cryo-transmission electron microscopy (cryo-TEM) supports the structural insight from hydrodynamic studies, particularly, in terms of the observed spherical structure of the formed micelles. We demonstrate experimentally that the micelle systems can be considered as solvent-permeable, hydrated spheres.

Nanoparticle Formulation Composition Analysis by Liquid Chromatography on Reversed-Phase Monolithic Silica.

Multifunctional nanoparticle (NP) formulations for medical purposes have already found their way toward envisaged translation. A persistent challenge of those systems is, next to NP size analysis, the compositional analysis of the NPs with the polymer as the matrix component and the encapsulated drug, particularly in a quantitative manner. Herein, we report the formulation of poly(lactic-co-glycolic acid) (PLGA) NPs by nanoprecipitation and the analysis of their integrity and size by dynamic light scattering (DLS) and scanning electron microscopy (SEM). Those NPs feature a variety of encapsulated drugs including the well-known ibuprofen (Ibu) as well as dexamethasone (Dex) and dexamethasone acetate (DexAce), with the latter being of potential interest for clinical treatment of SARS-CoV-2 patients. All those dissolved formulation compositions have been subjected to liquid chromatography on reversed-phase silica monolithic columns, allowing to quantitatively assess amounts of small molecule drug and NP constituting PLGA polymer in a single run. The chromatographically resolved hydrophobicity differences of the drugs correlated with their formulation loading and were clearly separated from the PLGA matrix polymer with high resolution. Our study identifies the viability of reversed-phase monolithic silica in the chromatography of both small drug molecules and particularly pharmapolymers in a repeatable and simultaneous fashion, and can provide a valuable strategy for analysis of diverse precursor polymer systems and drug components in multifunctional drug formulations.

PEtOxylated Interferon-α2a Bioconjugates Addressing H1N1 Influenza A Virus Infection.

Influenza A viruses (IAV), including the pandemic 2009 (pdm09) H1N1 or avian influenza H5N1 virus, may advance into more pathogenic, potentially antiviral drug-resistant strains (including loss of susceptibility against oseltamivir). Such IAV strains fuel the risk of future global outbreaks, to which this study responds by re-engineering Interferon-α2a (IFN-α2a) bioconjugates into influenza therapeutics. Type-I interferons such as IFN-α2a play an essential role in influenza infection and may prevent serious disease courses. We site-specifically conjugated a genetically engineered IFN-α2a mutant to poly(2-ethyl-2-oxazoline)s (PEtOx) of different molecular weights by strain-promoted azide-alkyne cyclo-addition. The promising pharmacokinetic profile of the 25 kDa PEtOx bioconjugate in mice echoed an efficacy in IAV-infected ferrets. One intraperitoneal administration of this bioconjugate, but not the marketed IFN-α2a bioconjugate, changed the disease course similar to oseltamivir, given orally twice every study day. PEtOxylated IFN-α2a bioconjugates may expand our therapeutic arsenal against future influenza pandemics, particularly in light of rising first-line antiviral drug resistance to IAV.

Ethoxy acetalated dextran-based nanocarriers accomplish efficient inhibition of leukotriene formation by a novel FLAP antagonist in human leukocytes and blood.

Leukotrienes are pro-inflammatory lipid mediators generated by 5-lipoxygenase aided by the 5-lipoxygenase-activating protein (FLAP). BRP-201, a novel benzimidazole-based FLAP antagonist, inhibits leukotriene biosynthesis in isolated leukocytes. However, like other FLAP antagonists, BRP-201 fails to effectively suppress leukotriene formation in blood, which limits its therapeutic value. Here, we describe the encapsulation of BRP-201 into poly(lactide-co-glycolide) (PLGA) and ethoxy acetalated dextran (Ace-DEX) nanoparticles (NPs), aiming to overcome these detrimental pharmacokinetic limitations and to enhance the bioactivity of BRP-201. NPs loaded with BRP-201 were produced via nanoprecipitation and the physicochemical properties of the NPs were analyzed in-depth using dynamic light scattering (size, dispersity, degradation), electrophoretic light scattering (effective charge), NP tracking analysis (size, dispersity), scanning electron microscopy (size and morphology), UV-VIS spectroscopy (drug loading), an analytical ultracentrifuge (drug release, degradation kinetics), and Raman spectroscopy (chemical attributes). Biological assays were performed to study cytotoxicity, cellular uptake, and efficiency of BRP-201-loaded NPs versus free BRP-201 to suppress leukotriene formation in primary human leukocytes and whole blood. Both PLGA- and Ace-DEX-based NPs were significantly more efficient to inhibit leukotriene formation in neutrophils versus free drug. Whole blood experiments revealed that encapsulation of BRP-201 into Ace-DEX NPs strongly increases its potency, especially upon pro-longed (≥ 5 h) incubations and upon lipopolysaccharide-challenge of blood. Finally, intravenous injection of BRP-201-loaded NPs significantly suppressed leukotriene levels in blood of mice in vivo. These results reveal the feasibility of our pharmacological approach using a novel FLAP antagonist encapsulated into Ace-DEX-based NPs with improved efficiency in blood to suppress leukotriene biosynthesis.

Reincarnation of the Analytical Ultracentrifuge: Emerging Opportunities for Nanomedicine.

The analytical ultracentrifuge (AUC) and the modern field of analytical ultracentrifugation found its inception approximately a century ago. We highlight the scope of its major experimental opportunities as a transport-based method, contemporary and up-and-coming investigation potential for polymers, polymer-drug conjugates, polymer assemblies, as well as medical nanoparticles. Special focus lies on molar mass estimates of unimeric polymeric species, self-assemblies in solution, and (co)localization of multicomponent systems in solution alongside the material-biofluid interactions. We close with present challenges and incentives for future research.

Drug-Induced Dynamics of Bile Colloids.

Bile colloids containing taurocholate and lecithin are essential for the solubilization of hydrophobic molecules including poorly water-soluble drugs such as Perphenazine. We detail the impact of Perphenazine concentrations on taurocholate/lecithin colloids using analytical ultracentrifugation, dynamic light scattering, small-angle neutron scattering, nuclear magnetic resonance spectroscopy, coarse-grained molecular dynamics simulations, and isothermal titration calorimetry. Perphenazine impacted colloidal molecular arrangement, structure, and binding thermodynamics in a concentration-dependent manner. At low concentration, Perphenazine was integrated into stable and large taurocholate/lecithin colloids and close to lecithin. Integration of Perphenazine into these colloids was exothermic. At higher Perphenazine concentration, the taurocholate/lecithin colloids had an approximately 5-fold reduction in apparent hydrodynamic size, heat release was less exothermic upon drug integration into the colloids, and Perphenazine interacted with both lecithin and taurocholate. In addition, Perphenazine induced a morphological transition from vesicles to wormlike micelles as indicated by neutron scattering. Despite these surprising colloidal dynamics, these natural colloids successfully ensured stable relative amounts of free Perphenazine throughout the entire drug concentration range tested here. Future studies are required to further detail these findings both on a molecular structural basis and in terms of in vivo relevance.

Overcoming the Necessity of a Lateral Aggregation in the Formation of Supramolecular Polymer Bottlebrushes in Water.

The assembly of supramolecular polymer bottlebrushes in aqueous systems is, in most cases, associated with a lateral aggregation of the supramolecular building blocks in addition to their axial stacking. Here, it is demonstrated that this limitation can be overcome by attaching three polymer chains to a central supramolecular unit that possesses a sufficiently high number of hydrogen bonding units to compensate for the increased steric strain. Therefore, a 1,3,5-benzenetrisurea-polyethylene oxide conjugate is modified with different peptide units located next to the urea groups which should facilitate self-assembly in water. For a single amino acid per arm, spherical micelles are obtained for all three tested amino acids (alanine, leucine, and phenylalanine) featuring different hydrophobicities. Only a slight increase in size and solution stability of spherical micelles is observed with increasing hydrophobicity of amino acid unit. In contrast, introducing two amino acid units per arm and thus increasing the number of hydrogen bonds per unimer molecule results in the formation of cylindrical structures, that is, supramolecular polymer bottlebrushes, despite a suppressed lateral aggregation. Consequently, it can be concluded that the number of hydrogen bonds has a more profound impact on the resulting solution morphology than the hydrophobicity of the amino acid unit.

Adjusting the length of supramolecular polymer bottlebrushes by top-down approaches.

Controlling the length of one-dimensional (1D) polymer nanostructures remains a key challenge on the way toward the applications of these structures. Here, we demonstrate that top-down processing facilitates a straightforward adjustment of the length of polyethylene oxide (PEO)-based supramolecular polymer bottlebrushes (SPBs) in aqueous solutions. These cylindrical structures self-assemble via directional hydrogen bonds formed by benzenetrisurea (BTU) or benzenetrispeptide (BTP) motifs located within the hydrophobic core of the fiber. A slow transition from different organic solvents to water leads first to the formation of µm-long fibers, which can subsequently be fragmented by ultrasonication or dual asymmetric centrifugation. The latter allows for a better adjustment of applied shear stresses, and thus enables access to differently sized fragments depending on time and rotation rate. Extended sonication and scission analysis further allowed an estimation of tensile strengths of around 16 MPa for both the BTU and BTP systems. In combination with the high kinetic stability of these SPBs, the applied top-down methods represent an easily implementable technique toward 1D polymer nanostructures with an adjustable length in the range of interest for perspective biomedical applications.

In Situ, Quantitative Assessment of Multifunctional Nanoscale Drug Delivery Systems in Human Serum.

The large volume and diversified nanomedicine market, undergoing a rapid growth, relies not only on the creation and applicative exploration of nanocarrier-based medicines showing significant potential, but in particular, demands a quantitative assessment of their physicochemical properties. In this study, we demonstrate the in situ assessment of multifunctional biodegradable nanoparticle (NP) entries as core components of nanoscale drug delivery systems (NDDSs) by making use of analytical ultracentrifugation (AUC). We determine and elucidate the following characteristics of NPs in NDDSs: NP density and size, targeting dye functionality, encapsulated and free drug, surfactant, and also NP drug release dynamics, quantitatively interconnected to NP degradation. In concept, we demonstrate this by multidetection AUC experiments at variable speed and time profiles. We could verify the quantitative and accurate nature of AUC for assessment of NDDSs, that is, also future nanomedicines. This concerns modeled and real life solution application formats such as cell culture media and human serum.

Analytical ultracentrifugation (AUC): a seminal tool offering multiple solutions.

Analytical ultracentrifugation (AUC) remains a highly versatile and widely applicable tool for the analysis of macromolecules and their interactions. The current state-of-the-art was demonstrated at a recent international meeting held in Glasgow, Scotland, in July 2017, the 23rd International Analytical Ultracentrifugation Workshop and Symposium. This special issue showcases the reports made at the meeting, which concerned the application of AUC to a wide range of topics in biochemical and polymer science including antibody and membrane protein characterisation, and protein-carbohydrate interactions. Presentations on development and testing of new instrumentation and methods of analysis were a particular feature of the meeting, including the optimisation of experimental protocols, and the latest optimised computational approaches to experimental simulation and the modelling of macromolecular structures.

Photocontrolled Release of Chemicals from Nano- and Microparticle Containers.

A benzoin-derived diol linker was synthesized and used to generate biocompatible polyesters that can be fully decomposed on demand upon UV irradiation. Extensive structural optimization of the linker unit was performed to enable the defined encapsulation of diverse organic compounds in the polymeric structures and allow for a well-controllable polymer cleavage process. Selective tracking of the release kinetics of encapsulated model compounds from the polymeric nano- and microparticle containers was performed by confocal laser scanning microscopy in a proof-of-principle study. The physicochemical properties of the incorporated and released model compounds ranged from fully hydrophilic to fully hydrophobic. The demonstrated biocompatibility of the utilized polyesters and degradation products enables their use in advanced applications, for example, for the smart packaging of UV-sensitive pharmaceuticals, nutritional components, or even in the area of spatially selective self-healing processes.

Hydrodynamic Analysis Resolves the Pharmaceutically-Relevant Absolute Molar Mass and Solution Properties of Synthetic Poly(ethylene glycol)s Created by Varying Initiation Sites.

The solution behavior originating from molecular characteristics of synthetic macromolecules plays a pivotal role in many areas, in particular the life sciences. This situation necessitates the use of complementary hydrodynamic analytical methods as the only means for a complete structural understanding of any macromolecule in solution. To this end, we present a combined hydrodynamic approach for studying in-house prepared, low dispersity poly(ethylene glycols)s (PEGs), also known as poly(ethylene oxide)s (PEOs) depending on the classification used, synthesized from varying initiation sites by the living anionic ring opening polymerization. The series of linear PEGs in the molar mass range of only a few thousand to 50 000 g mol-1 have been studied in detail via viscometry and sedimentation-diffusion analysis by analytical ultracentrifugation. The obtained estimations for intrinsic viscosity, diffusion coefficients, and sedimentation coefficients of the macromolecules in the solution-based analysis clearly showed self-consistency of the followed hydrodynamic approach. This self-consistency is underpinned by appropriate and physically sound values of hydrodynamic invariants, indicating adequate values of derived absolute molar masses. The classical scaling relations of Kuhn-Mark-Houwink-Sakurada of all molar-mass dependent hydrodynamic estimates show linear trends, allowing for interrelation of all parametric macromolecular characteristics. Differences among these are ascribed to the observation of α-end and chain-length dependent solvation of the macromolecules, identified from viscometric studies. This important information allows for analytical tracing of variations of scaling relationships and a physically sound estimation of hydrodynamic characteristics. The demonstrated self-sufficient methodology paves an important way for a complete structural understanding and potential replacement of pharmaceutically relevant PEGs by alternative macromolecules offering a suite of similar or tractably distinct physicochemical properties.

Determination of ω-end functionalities in tailored poly(2-alkyl-2-oxazoline)s by liquid chromatography and mass spectrometry.

The in-depth analytical characterization of polymers, in particular regarding intended biomedical applications, is becoming increasingly important to elucidate their structure-property relationships. Specifically, end group analysis of e.g. polymers featuring a 'stealth effect' towards the immune system is of particular importance because of their use in coupling reactions to bioactive compounds. Herein, we established a liquid chromatography (LC) protocol to analyse bicyclo[6.1.0]nonyne-functionalized poly(2-alkyl-2-oxazoline)s (POx)s as promising functional polymers that can be applied in strain-promoted click reactions. This work involved the synthesis of poly(2-methyl-2-oxazoline) (PMeOx) and poly(2-ethyl-2-oxazoline) (PEtOx) by living cationic ring-opening polymerization (CROP) with different molar masses ranging from 2 up to 17.5 kDa and, to our knowledge, the first liquid chromatographic analysis of PMeOx. The developed analytical protocol enables the quantitative determination of post-polymerization reaction sequences with respect to the conversion of the ω-end groups. All synthesized polymers were straightforwardly analysed on a C18-derivatized silica monolithic column under reversed-phase chromatographic conditions with a binary mobile phase gradient comprising a mixture of acetonitrile and water. Subsequent mass spectrometry of collected elution fractions enabled the confirmation of the desired ω-end group functionalities and the identification of synthetic by-products.

PEG-Lipid-PLGA Hybrid Particles for Targeted Delivery of Anti-Inflammatory Drugs.

Hybrid nanoparticles (HNPs) were designed by combining a PLGA core with a lipid shell that incorporated PEG-Lipid conjugates with various functionalities (-RGD, -cRGD, -NH2, and -COOH) to create targeted drug delivery systems. Loaded with a neutral lipid orange dye, the HNPs were extensively characterized using various techniques and investigated for their uptake in human monocyte-derived macrophages (MDMs) using FC and CLSM. Moreover, the best-performing HNPs (i.e., HNP-COOH and HNP-RGD as well as HNP-RGD/COOH mixed) were loaded with the anti-inflammatory drug BRP-201 and prepared in two size ranges (dH ~140 nm and dH ~250 nm). The HNPs were examined further for their stability, degradation, MDM uptake, and drug delivery efficiency by studying the inhibition of 5-lipoxygenase (5-LOX) product formation, whereby HNP-COOH and HNP-RGD both exhibited superior uptake, and the HNP-COOH/RGD (2:1) displayed the highest inhibition.

Assessing the nanoscale structure and mechanical properties of polymer monoliths used for chromatography.

Concerning polymeric monolithic materials utilized in separation science, the structural and mechanical characteristics from the nanoscopic to the macroscopic scale remain of great interest. Suitable analytical tools are urgently required to understand the polymer monolith's constituent structure, particularly in the case of nanoscale polymer properties that tend to develop gel porosity in contact with a mobile phase ultimately affecting the chromatographic performance. Herein described are our first findings from a characterization of commercially available analytical polymer monoliths based on styrene/divinylbenzene and methacrylate chemistries utilizing confocal Raman spectroscopy imaging and atomic force microscopy (AFM). Confocal Raman spectroscopy can be used to generate a three-dimensional representation of monoliths in both dry state and in contact with solvent. AFM force-indentation measurements on individual cross-sectioned globular features permit detailed assessment of mechanical properties of the stationary phase. This approach allowed so far unprecedented insight and identification of a heterogeneous cross-link density distribution of polymer material within individual globular features on a submicrometer scale.

All-Aqueous, Surfactant-Free, and pH-Driven Nanoformulation Methods of Dual-Responsive Polymer Nanoparticles and their Potential use as Nanocarriers of pH-Sensitive Drugs.

All-aqueous, surfactant-free, and pH-driven nanoformulation methods to generate pH- and temperature-responsive polymer nanoparticles (NPs) are described. Copolymers comprising a poly(methyl methacrylate) (PMMA) backbone with a few units of 2-(dimethylamino)ethyl methacrylate (DMAEMA) are solubilized in acidic buffer (pH 2.0) to produce pH-sensitive NPs. Copolymers of different molar mass (2.3-11.5 kg mol-1 ) and DMAEMA composition (7.3-14.2 mol%) are evaluated using a "conventional" pH-driven nanoformulation method (i.e., adding an aqueous polymer solution (acidic buffer) into an aqueous non-solvent (basic buffer)) and a robotized method for pH adjustment of polymer dispersions. Dynamic light scattering, zeta-potential (ζ), and sedimentation-diffusion analyses suggest the formation of dual-responsive NPs of tunable size (from 20 to 110 nm) being stable for at least 28 days in the pH and temperature intervals from 2.0 to 6.0 and 25 to 50 °C, respectively. Ultraviolet-visible spectroscopic experiments show that these NPs can act as nanocarriers for the pH-sensitive dipyridamole drug, expanding its bioavailability and potential controlled release as a function of pH and temperature. These approaches offer alternative strategies to prepare stimuli-responsive NPs, avoiding the use of harmful solvents and complex purification steps, and improving the availability of biocompatible polymer nanoformulations for specific controlled release of pH-sensitive cargos.

Nanoparticle sizing in the field of nanomedicine: Power of an analytical ultracentrifuge.

Hydrodynamic and light scattering methods are urgently required for accurate characterization of nanoparticles (NPs) in the field of nanomedicine to unveil their sizes and distributions. A fundamental characterization approach in the field of nanomedicines is, next to standard batch dynamic light scattering (DLS) and increasingly more applied (asymmetrical flow) field-flow fractionation (FFF) coupled to multi-angle laser light scattering (MALLS), the utilization of an analytical ultracentrifuge (AUC). Here, we demonstrate the power of an AUC in comparison to batch DLS and FFF-MALLS to decipher, in detail, the size and dispersity of pharma-relevant, commercial and in-house prepared soft matter NPs, suitable for life science applications. In this study, size and dispersity of poly(lactic-co-glycolic acid) (PLGA) NPs and in-house prepared NPs, consisting of the commercially available pharmapolymer Eudragit® E or of a polymer of similar composition synthesized via reversible addition fragmentation chain transfer (RAFT) polymerization, were investigated. Simultaneously, an insight on the presence of the utilized surfactant on the NP formulations, which is usually limited with other techniques, could be achieved by multi-speed experiments with the AUC in one experimental setting. While the repeatability and ruggedness of observations with modern AUC instruments of the newest generation is demonstrated, the results are further underpinned by the classical relations of hydrodynamics. Investigations aiming at hydrodynamic diameters (from DLS) and radii of gyration (from FFF-MALLS) are critically discussed and compared to the repeatable and rugged investigations by an AUC. The latter is proven to provide a self-sufficient experimental approach for NP characterization in the field of nanomedicine based on absolute principles, compares well to FFF-MALLS, and can unravel issues in NP sizing that arise when more common techniques, such as DLS, are used.

Polymer selection impacts the pharmaceutical profile of site-specifically conjugated Interferon-α2a.

Conjugation of poly(ethylene glycol) (PEG) to biologics is a successful strategy to favorably impact the pharmacokinetics and efficacy of the resulting bioconjugate. We compare bioconjugates synthesized by strain-promoted azide-alkyne cycloaddition (SPAAC) using PEG and linear polyglycerol (LPG) of about 20 kDa or 40 kDa, respectively, with an azido functionalized human Interferon-α2a (IFN-α2a) mutant. Site-specific PEGylation and LPGylation resulted in IFN-α2a bioconjugates with improved in vitro potency compared to commercial Pegasys. LPGylated bioconjugates had faster disposition kinetics despite comparable hydrodynamic radii to their PEGylated analogues. Overall exposure of the PEGylated IFN-α2a with a 40 kDa polymer exceeded Pegasys, which, in return, was similar to the 40 kDa LPGylated conjugates. The study points to an expanded polymer design space through which the selected polymer class may result in a different distribution of the studied bioconjugates.

Porous polymer monoliths for small molecule separations: advancements and limitations.

Porous polymer monoliths are considered to be one of the major breakthroughs in separation science. These materials are well known to be best suited for the separation of large molecules, specifically proteins, an observation most often explained by convective mass transfer and the absence of small pores in the polymer scaffold. However, this conception is not sufficient to explain the performance of small molecules. This review focuses in particular on the preparation of (macro)porous polymer monoliths by simple free-radical processes and the key events in their formation. There is special focus on the fluid transport properties in the heterogeneous macropore space (flow dispersion) and on the transport of small molecules in the swollen, and sometimes permanently porous, globule-scale polymer matrix. For small molecule applications in liquid chromatography, it is consistently found in the literature that the major limit for the application of macroporous polymer monoliths lies not in the optimization of surface area and/or modification of the material and microscopic morphological properties only, but in the improvement of mass transfer properties. In this review we discuss the effect of resistance to mass transfer arising from the nanoscale gel porosity. Gel porosity induces stagnant mass transfer zones in chromatographic processes, which hamper mass transfer efficiency and have a detrimental effect on macroscopic chromatographic dispersion under equilibrium (isocratic) elution conditions. The inherent inhomogeneity of polymer networks derived from free-radical cross-linking polymerization, and hence the absence of a rigid (meso)porous pore space, represents a major challenge for the preparation of efficient polymeric materials for the separation of small molecules.

Separation of adrenergic amines in Citrus aurantium L. var. amara by capillary electrochromatography using a novel monolithic stationary phase.

This manuscript reports on the use of capillary electrochromatography for the determination of tyramine, (±) synephrine, and (±) octopamine, the major alkaloids in bitter orange peel. A novel methacrylate-based monolithic stationary phase was prepared by UV-photopolymerization in 100 µm id fused-silica capillaries. It facilitated the quantitative assessment of alkaloids with a mobile phase comprising aqueous 10 mM ammonium acetate in ACN and isopropanol. Applied voltage and temperature were 25 kV and 25°C, and samples were injected in electrokinetic mode. The method reported herein revealed adequate sensitivity (LOD ≤0.6 µg/mL), repeatability (σrel ≤4.1%), accuracy (recovery rates between 95.2 and 102.2%), and precision (intra-day variation ≤5.7%, inter-day variation ≤4.1%). The application of the CEC assay on C. aurantium var. amara plant material and dietary supplements, which usually are advertised for slimming properties, indicated that synephrine (0.17-0.82%) is the dominant alkaloid.