The ICH guideline Q14 on analytical procedure development underlines the importance of science and risk-based methods for the evaluation of the quality of medicines. Ultimately, a pharmaceutical company, the sponsor, is responsible that the analytical method is fit-for-purpose during routine use throughout its lifecycle. Part of the analytical procedure control strategy is the responsibility to assure availability of critical materials of the analytical method. For capillary zone electrophoresis (CZE) methods, the background electrolyte (BGE) composition is a key and critical material. In this study, we investigated whether key ingredients of the ε-aminocaproic acid (eACA) CZE (eACA-CZE) method for monoclonal antibodies can be replaced by structurally related chemicals. The complex heterogeneity patterns are compared, as well as the reportable results as the percentage main, acidic and basic peaks. Overall, the results underline the ruggedness of the eACA-CZE method and provide alternative options to eACA and triethyltetramine (TETA), in case there are quality or supply issues, thus de-risking and safeguarding release and stability studies for therapeutic mAbs.

Green analytical chemistry (GAC) aims to achieve faster, safer, accurate, and sustainable analysis by minimizing the hazardous impact of organic solvents on the environment. Here, the focus will be on impurity profiling of pharmaceuticals. This can be done by employing green chromatographic techniques using eco-friendly solvents, advanced spectroscopic approaches, microextraction, and miniaturization techniques. Pharmaceutical analysis's greenness can be evaluated using various assessment tools and the advanced analytical greenness metric (AGREE). This review comparatively discusses all these tools by highlighting their eco-friendly nature, methodology, adherence to the 12 major principles of green chemistry, and validation of their results. Furthermore, state-of-the-art case studies utilizing green chemistry principles for impurity profiling of pharmaceuticals have also been discussed, along with their employed techniques and observations, thus offering insights into successful implementation. Still, cost, affordability, scalability, and reproducibility vary across techniques, with, for example, supercritical fluid chromatography (SFC) showing more substantial potential than nuclear magnetic resonance (NMR). Greenness metrics also lack global harmonization, limiting comparability. Challenges like stringent regulatory frameworks and a lack of standardized global policies should be overcome by integrating artificial intelligence (AI) and machine learning (ML), as well as more interdisciplinary collaboration among associated professionals, for the widespread adoption of efficient, sustainable, and eco-friendlier pharmaceutical analysis techniques. For AI/ML, trustworthiness will require validation and regulatory alignment to ensure they complement rather than replace existing protocols.

Siponimod fumarate (SIP), a potent selective S1P receptor modulator, has emerged as a critical therapeutic agent in the treatment of multiple sclerosis. Recognizing the increasing regulatory demands for robust impurity profiling and method reliability, this study reports the development and optimization of an HPLC method for the simultaneous determination of SIP and its related impurities in both bulk drug substance and tablet dosage forms. The method was developed within an Analytical Quality by Design (AQbD) framework, guided by ICH Q14 principles, ensuring a systematic and risk-based approach throughout the analytical lifecycle. Chromatographic separation necessary for resolving critical impurities was achieved on an XSelect HSS T3 column (150 mm × 4.6 mm, 3.5 µm) using a stepped gradient elution program with 0.1% perchloric acid in water and acetonitrile as the mobile phases. Optimal separation conditions, identified through the AQbD process to meet stringent performance criteria, were determined at a column temperature of 42.5°C, a flow rate of 1.4 mL min, and UV detection at 212 nm. The method performance was rigorously evaluated through accuracy profiles, confirming both its precision and trueness across the targeted concentration range. In parallel, as part of a holistic method characterization, environmental sustainability was assessed using comprehensive greenness metrics, whereas its practical applicability was further substantiated using the Blue Applicability Grade Index (BAGI) and the Red-Green-Blue 12 (RGB12) algorithms. This approach not only bridges the gap created by the absence of an official pharmacopoeial monograph for SIP but also offers a robust, well-characterized, and sustainable platform for pharmaceutical quality control, aligning method development with both regulatory performance needs and environmental awareness.

Backscatter interferometry (BSI) is a refractive index detection method for capillary electrophoresis that is inexpensive, flexible, and easily miniaturized. Interestingly, unlike most detectors that respond exclusively to analyte concentration, the BSI signal is sensitive to both refractive index (analyte concentration) and the separation voltage. The latter is linked to zone conductivity and leads to improved BSI signals and lower detection limits with increasing field strengths. Enhanced BSI signals can also be generated using a photothermal mechanism, where resonantly excited analytes release heat into their surroundings to increase the BSI signal amplitude. Both voltage-based and photothermal signal enhancement mechanisms can lead to a change in the polarity of the BSI signal, which can be either positive or negative depending on the specific analyte, its concentration, and the separation conditions. Here, we show that this leads to a significant increase in apparent peak efficiency. At the transition in peak polarity, both mechanisms result in over a 10-fold increase in apparent peak efficiency, improving from approximately 10 plates/m to over a million plates/m. Simultaneously measured BSI and fluorescence electropherograms confirm that the efficiency increase is unique to the BSI signal and not due to changes in zone dispersion, and can be tuned to optimize separation resolution. The origin of the efficiency increase is discussed in terms of the refractive index and zone conductivity contributions to the BSI signal.

Substandard and falsified medicines pose a significant public health threat, particularly in low-income countries. Ensuring pharmaceutical quality is crucial to mitigate risks associated with ineffective and harmful medications. Among others, developing and implementing robust and cost-effective analytical methods is an important and quick strategy for ensuring the quality of medicines. This study aimed to develop a robust and cost-effective capillary zone electrophoresis method with UV detection for insulin analysis in active pharmaceutical ingredient and formulations. A multilayer capillary coated with polybrene and poly(sodium 4-styrenesulfonate) improved repeatability. The method was optimized by systematically evaluating running buffer composition, pH, ionic strength, and voltage, achieving optimal separation with a 60 mM phosphate buffer at pH 8.0. It demonstrated excellent precision, accuracy, linearity, and robustness. Application of the method to insulin commercial samples verified compliance with pharmacopoeial standards. The method could be a reliable and accessible alternative for quality control of insulin in resource-limited settings, supporting efforts to combat substandard pharmaceuticals and protect public health. Moreover, the method aligns with green chemistry principles, as it eliminates the need for organic solvents, either as solvent or as a component of the running buffer.

High-performance capillary electrophoresis (CE) has been widely applied in the analysis of organic acids, especially monocarboxylic acids (MAs), but no studies on the CE analysis of dissociated states of MAs have been reported. Here, 15 MAs were analyzed by the newly developed universal interface-induced current detector (IICRD). Two current signal peaks were observed in current electrophoretograms (CR-EGs), whereas only one peak with low sensitivity can be obtained by diode array detector (DAD). The current signal peaks of MAs were accurately identified by adding a charge-neutral marker and calculating by pK. The qualitative analysis indicated that the two current signal peaks of MAs were charge-neutral forms [MA] and monovalent anions [MA] in order. Quantitative analysis showed that CE-IICRD can enhance the sensitivity of MA analytes to 40 µM in limit of detection (LOD), with a linear range from 10 to 10 M. Furthermore, interactions between different dissociated states of MAs and monocarboxylate transporter 1 (MCT1) were studied by combined application of nonimmobilized cell CE (NICCE) method for the first time. The binding kinetic parameters and mole number of MCT1 on cell membranes can also be obtained. Besides, competitive binding experiments proved that BAY-8002 (MCT1-specific inhibitor) and lactic acid shared the same binding site. CE-IICRD provides a new method to reveal the interaction between different dissociated forms of MAs or other biomolecules and essential receptors.

CRISPR-Cas9-targeted sequencing can enrich DNA regions of interest by directing the Cas9 protein to bind and cleave specific DNA sequences via single-guide RNA (sgRNA). It is interesting to explore the efficacy of using CRISPR-Cas9-targeted nanopore sequencing (referred to as Cas9-seq), a polymerase chain reaction (PCR)-free workflow, for forensic short tandem repeats (STR) profiling, and to compare it with the amplification-based approach. In this pilot study, we constructed a Cas9-seq method for profiling seven STR loci, including D18S51, FGA, TPOX, D16S539, vWA, CSF1PO, and TH01. With 3 µg DNA inputs from human NA12878 and 293T cell lines, we achieved 643.45- and 468.34-fold enrichment ratios of the sgRNA-targeted regions by using Cas9-seq, respectively. Compared to nanopore sequencing of PCR amplicon products (amplicon-seq) of the ForenSeq DNA Signature Prep kit, the Cas9-seq reads had an ultralow strand bias. However, surprisingly, Cas9-seq did not show advantages in allele balance and had higher noise in the reads. At the seven STR loci for the two samples, both Cas9-seq and amplicon-seq had three genotyping errors. Additionally, there were no false-positive single-nucleotide polymorphisms (SNPs) introduced by Cas9-seq, whereas amplicon-seq produced three. In sum, we conclude that the PCR-free Cas9-seq might not be favorable for forensic STR genotyping.

This review presents a comprehensive overview of the developments and applications of high-performance capillary and microchip electromigration methods (zone electrophoresis in a free solution or in sieving media, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography, and electrochromatography) for analysis, microscale isolation, and physicochemical and biochemical characterization of peptides in the period from 2023 up to ca. the middle of 2025. Advances in the exploration of electromigration properties of peptides and various aspects of their analysis, such as sample preparation, sorption suppression, EOF regulation, and detection, are described. New developments in the particular CE methods are presented, and several types of their applications are reported. They include qualitative and quantitative analysis of synthetic or isolated peptides, determination of peptides in complex biomatrices, peptide profiling of biofluids and tissue extracts, and monitoring of chemical and enzymatic reactions and physicochemical changes of peptides. They also deal with amino acid and sequence analysis of peptides, peptide mapping of proteins, separation of stereoisomers of peptides, and their chiral analyses. In addition, micropreparative separations and physicochemical and biochemical characterizations of peptides and their interactions with other (bio)molecules by the above CE methods are described.

Brucellosis is a neglected zoonotic disease that continues to impact global public health and livestock economies, particularly in regions with limited diagnostic infrastructure. Its causative agent, Brucella abortus, is difficult to detect due to its intracellular lifestyle and the nonspecific symptoms it causes in humans. This study demonstrates the experimental application of dielectrophoresis (DEP) in a microfluidic device for the selective manipulation of polystyrene beads and inactivated B. abortus bacteria. By tuning the frequency and medium conductivity, reliable combined negative dielectrophoretic (nDEP) and hydrodynamic flow responses were achieved, leading to the deflection of bacterial cells across the microchannel within a critical vertical window for particle control. Distinct particle trajectories were observed under varying electric field conditions, confirming effective separation without the need for labels or biochemical markers, except for visual validation. This label-free strategy enables rapid sample processing and has the potential to be integrated into portable platforms for on-site diagnostics. The results highlight the feasibility of DEP-based approaches for pathogen separation and support their future implementation in brucellosis surveillance and point-of-care testing.

Dielectrophoresis (DEP) is a powerful tool for manipulating particles using non-uniform electric fields. This study combines numerical simulations and experiments to investigate crossover frequencies (COFs) for micro- and nanoparticles in a 3D microfluidic device with circular traps. MATLAB simulations revealed an inverse relationship between particle size and COF. For microparticles with diameters of 1.03, 2.27, 4.42, and 6.83 µm, the COFs were calculated as 769.10, 352.76, 183.96, and 120.51 kHz, respectively. For nanoparticles measuring 50, 170, and 500 nm, the corresponding COFs were 15.6, 4.62, and 1.57 MHz. These results closely matched experimental data. Notably, additional low-frequency pseudo-COFs emerged in experiments for nanoparticles ranging from 2 to 8 kHz (50 nm), 10 to 50 kHz (170 nm), and 40 to 100 kHz (500 nm). These frequencies proportionally increased with nanoparticle size and corresponded to unexpected negative DEP (nDEP)-like behavior under positive DEP (pDEP) conditions. This effect is attributed to low-frequency alternating current electroosmosis (ACEO), which dominates the DEP response of the nanoparticles smaller than 1 µm. These findings demonstrate strong agreement between numerical simulations and experimental results while also revealing the limitations of traditional models in predicting nanoparticle behavior under DEP. We expect that these results can also be applied to the manipulation of various bioparticles.

Electrokinetic phenomena play a vital role in the study of colloidal nanoparticles, offering significant insights and applications across a wide range of fundamental research and practical uses. It is crucial to recognize the extensive research on various types of nanomaterials, including polymer latexes, quantum dots, and biomolecules. However, there is a significant gap in the study of magnetic systems. Such materials have immense potential and can greatly benefit from both magnetophoretic and electrophoretic techniques. In this work, the electrophoretic behavior of water-based magnetic fluids was investigated, focusing on how additional magnetic field exposure affects their properties. The studies conducted utilized magnetic measurements that emerged from the presence of colloidal particles within the examined systems, which exhibited both charge and magnetic moment. The magnetic susceptibility and magnetization of colloidal particles precipitate formed on one of the electrodes were measured. The thickness of the formed precipitate on the electrode can be confidently estimated through micrometric measurements as well as by analyzing its magnetic susceptibility during electrophoresis. A formula for calculating the electrophoretic velocity based on the results of magnetic measurements was obtained. Estimates of zeta potential and charge of colloidal particles were carried out. The electrophoresis process in these systems can be effectively regulated by an inhomogeneous magnetic field, leading to complete compensation.

This work investigates electrokinetically driven protein transport in open microfluidic devices with microwells featuring axial shape variations. The results indicate that protein propagation, which is lysed at the surface, depends on two key parameters: the electrical potential ratio , and the geometric curvature of the microwell. The concave microwell configuration presents the best outcome due to the emergence of a transverse velocity component that confines the cell within the microwell. Lastly, protein concentration can be improved when the negative microwell geometric slope exhibits nondifferentiable behavior (e.g., edges or fractal geometries), while a higher zeta potential can broaden the influence of the Stern layer.

This work focuses on the separation of oligonucleotides (ONs) in a free solution by capillary electrophoresis-mass spectrometry (CE-MS). Specifically, we evaluated a combination of separation in acidic background electrolytes (BGEs), nanospray ionization, and time-of-flight mass spectrometry in positive mode. A mixture of synthetic ONs, ranging in length from 15 to 78 nt, was employed as the test compounds. The key to a good separation selectivity of ONs lies in the different protonation of individual nucleobases. To this end, we assessed the acidity constant (pK) of the nucleobases in nucleotides experimentally as 3.3 for adenine, 4.4 for cytosine, 2.5 for guanine, and < 2 for thymine. From a set of separations in the 2-9 pH range, it was found that optimum peak shape and resolution are achieved in the interval of acidic pH 2-2.5. The BGE can be conveniently composed of either formic acid (FA) or a combination of ammonium hydroxide and FA. Nanospray ionization provided ions with charge numbers ranging from +4 to +8, proportional to the length of the ON. For short sequences, sheath liquid (SL) comprising 0.5%-1% (v/v) FA + 20% (v/v) methanol was sufficient in order to generate ions in positive mode MS, whereas a stronger SL of 5% (v/v) FA + 20% (v/v) methanol was required for longer ON sequences of approximately > 40 nt.

Lipid nanoparticles (LNPs) are widely used for the delivery of nucleic acid (NAs), most notably in gene therapy and messenger ribonucleic acid (mRNA)-based vaccines. Understanding their physicochemical properties is essential, yet current analytical approaches often fall short in capturing their complexity. Here, we introduce an analytical strategy using capillary zone electrophoresis (CZE) and pressure-driven Taylor dispersion (TD) analysis beside the combination of both separation principles. This novel separation mode of electrophoretic TD or electrohydrodynamic coupling (termed here as eTD) can be used to characterize deoxyribonucleic acid (DNA)-loaded LNP formulations using standard capillary electrophoresis (CE) instrumentation. eTD is a new separation approach that combines electrophoretic and hydrodynamic movement in micro-scaled capillaries for the analysis of drug carriers as LNPs. Focusing on critical quality attributes (CQAs), TD provided information on the hydrodynamic radius of LNPs and the distribution of NAs across different chemical environments. CZE enabled the estimation of ζ-potential and localization of DNA within distinct particle populations. The novel eTD mode offers deeper insight into LNP structure and morphological aspects, yielding characteristic profiles for individual formulations and revealing the presence of unencapsulated DNA. To contextualize LNP measurements, we also analysed free NAs and their mixtures with LNPs under identical conditions. The method distinguished between encapsulated and unencapsulated species, revealing individual electrophoretic and dispersion profiles for single-stranded mRNA and double-stranded DNA. These findings demonstrate the potential of capillary techniques for the advanced physicochemical characterization of NA-loaded LNPs. Further investigations are warranted to expand their analytical utility and deepen our understanding of LNP structural features.

Current Y-chromosomal single-nucleotide polymorphism (Y-SNP) detection technologies in forensic genetics often rely on bulky equipment, complex procedures, and lack adaptability to field conditions. To address these limitations, we developed an 89-plex microfluidic Y-SNP system comprising a disposable DNA extraction/amplification chip and a capillary electrophoresis chip. Using in situ lyophilization, the reagents are stabilized for long-term storage. Integrated with a portable device, the system enables a fully automated "sample-in-answer-out" workflow and delivers complete 89-locus Y-SNP genotyping within 139 min. The system includes two panels-AIYSNP42 for global high-frequency haplogroups and AIYSNP47 for East Asian O-haplogroup subclades-designed on the basis of the International Society of Genetic Genealogy (ISOGG) phylogenetic tree for multi-level resolution. Validation showed a detection sensitivity of 2.5 ng of DNA, with a 93.6% genotyping success rate across 94 forensic samples. It maintained performance under environmental inhibitors (humic acid ≤ 100 ng/µL, hemin ≤ 300 µM, indigo ≤ 15 mM) and moderate UV-induced DNA degradation. The system demonstrated excellent reproducibility (coefficient of variation, CV < 0.5%) and reliably detected male DNA in mixtures (≥2% in male-female, ≥33% in male-male). This microfluidic system reduces the reliance on the need for conventional laboratory workflows and supports rapid, on-site Y-SNP analysis for pedigree tracing, ancestry inference, and mixture interpretation.

This study investigates dynamic electrohydrodynamic (EHD) interactions between two identical colloidal microspheres in rotating electric fields using a fully coupled three-dimensional transient model. Long-range dielectrophoretic (DEP) attraction drives radial convergence; upon near-contact, tangential EHD sliding further induces asynchronous co-field orbital revolution. Crucially, individual electrorotation (ER) not only retains its original direction but also maintains a stable rate-with the rate deviating by <5% from that of isolated particles, matching single-particle behavior. High-frequency DEP force polarity reversal establishes stable noncontact equilibria via short-range repulsion. Spectral analyses reveal collective dynamics (radial mobility, orbital motion) stem from rotating electric field-mediated gap modulation rather than altered particle polarization. This dynamic decoupling-governed by the Kramers-Kronig relationship between real (radial DEP) and imaginary (rotational ER) components of polarizability-enables independent positional and rotational control, opening new avenues for noncontact colloidal manipulation in microfluidic mixers and dynamically reconfigurable active matter systems, where conventional DEP-based approaches are limited by coupled dynamics.

We demonstrate a gel-free electrophoretic separation of Sanger sequencing fragments up to 782 bases in length using nonionic wormlike micelles as drag-tags in micelle-tagging electrophoresis (MTE). This is an increase of 280 bases over previous MTE methods and a nearly three-fold improvement over end-labeled free-solution electrophoresis (ELFSE) methods that use covalently attached drag-tags. For MTE, C18 alkane groups are attached to primers prior to their enzymatic extension. This alkane group provides a binding site for wormlike micelles composed of CiEj-type nonionic surfactants in the running buffer. Transient attachment of micelles to the C18 alkane group provides a highly uniform drag, equal to that of an ssDNA fragment 309 bases long. To account for slight mobility differences among the BigDye chain terminators, we developed a two-parameter time-shifting procedure to align the electropherograms for each termination chemistry. The increase in read length for this low-viscosity buffer (2.1 cP) is attributed to the alignment procedure, the large yet uniform drag, and the small degree of adsorption-based band broadening.

Three-dimensional (3D) printing has revolutionized manufacturing by enabling the rapid fabrication of complex structures, yet conventional 3D techniques remain constrained by inherent limitations in resolution, speed, and multi-material integration. To address these challenges, emerging approaches such as microfluidic-assisted and field-assisted additive manufacturing have been developed to enhance the capabilities and versatility of the method. Microfluidic-assisted 3D printing leverages controlled flow patterns for material deposition and control, material gradient formation, and advanced polymerization processes. Field-assisted methods, including electric-, acoustic-, and interface-assisted approaches, directly manipulate materials during printing to enable advanced functionalities and material properties. This review summarizes the latest advancements in microfluidic- and field-assisted 3D printing, highlighting their unique advantage in overcoming current 3D printing limitations and their potential to drive innovation in applications ranging from biomedical devices to functional materials development.

A green method by capillary electrophoresis (CE) is described for the first time for the determination of dapagliflozin (DAPA), an oral hypoglycemic drug approved for the treatment of Type 2 diabetes mellitus. The effects of different analytical conditions were evaluated, including the concentration and pH of the background electrolyte (BGE), sample injection time, applied voltage, as well as capillary temperature. The method was validated by establishing the linearity, intra- and interday precisions (relative standard deviation, RSD%), accuracy, and robustness. The analytical procedure was linear in the range of 50-175 µg mL (R > 0.999), with the limit of detection (LOD) and limit of quantitation (LOQ) of 6.2 and 18.8 µg mL, respectively. Precision had an intraday RSD of 2.55% and an interday RSD of 2.52%. The average recovery rates for the pharmaceutical samples ranged from 101.22% to 104.63%, with an RSD of 0.88%. Additionally, the CE method was compared to a high-performance liquid chromatography (HPLC) method for quantifying DAPA, and their green profiles were assessed by the Analytical Greenness Metric (AGREE), confirming the eco-friendliness of the CE technique. The methodology is suitable for determining DAPA in tablets; CE provides a greener alternative due to low-cost analysis using fewer organic solvents and minimizing waste generation.

In this study, ion chromatography (IC) methods were developed and validated for the determination of sodium, potassium, phosphate, and sorbitol in phosphate syrup. For the analysis of cations, an IonPac CS16 column was utilized, with a mobile phase of 50 mM methanesulfonic acid and a flow rate of 0.5 mL/min. For the analysis of phosphate and sorbitol, an IonPac AS19 column was employed, using a flow rate of 1.0 mL/min and mobile phases of 50 and 20 mM NaOH, respectively. In the validation tests, sensitivity was assessed on the basis of the signal-to-noise ratio, with the limit of detection for all analytes being below 0.001 mM. The linearity curves for all analytes exhibited determination coefficients greater than 0.999, indicating excellent linearity. The relative standard deviation (RSD%) for both inter-day and intra-day precision was not more than 1%. Accuracy, expressed as recovery (%), ranged from 98% to 101% for all ions. The validation of these methods demonstrated their reliability for the measurement of these four analytes. Furthermore, the stability of the syrup was evaluated over 6 months at room temperature (25°C). The results indicated that the phosphate syrup remained stable under these conditions, with the analyte contents staying close to 100%.

A novel DC-dielectrophoresis (DEP) method employing a tunable insulating microdroplet for the continuous sorting of microtargets is presented in this article. To induce the dielectrophoretic effect, a DC electric voltage is applied via the microdroplet through the microchannel to induce the gradient of the inhomogeneous electric field. When passing through the gap between the microdroplet and the channel where there is the strongest nonuniformity of the electric field, the microparticles experience the DEP effects, and their trajectories shift. The effects of the gap spacing and the applied voltage on the distribution of the electric field gradient and the effect of the flow rate on the particle trajectory were analyzed numerically. On the basis of theoretical analysis, a tunable microdroplet-based microfluidic chip was fabricated, and the experimental system platform centered on the tunable droplet chip was constructed. Experiments were conducted to demonstrate the sorting of 5 and 10 µm polystyrene microparticles by adjusting the joint gap distance, flow rate, and applied voltage. The experimental results were in good agreement with the numerical simulation, which proved the feasibility of using microdroplet to serve as tunable insulator for the manipulation and separation of microtargets.