Partition coefficient screening - An effective approach for finding the best conditions for byproduct removal as demonstrated by a bispecific antibody purification case.

In recombinant protein purification, differences in isoelectric point (pI)/surface charge and hydrophobicity between the product and byproducts generally form the basis for separation. For bispecific antibodies (bsAbs), in many cases the physicochemical difference between product and byproducts is subtle, making byproduct removal considerably challenging. In a previous report, with a bsAb case study, we showed that partition coefficient (Kp) screening for the product and byproducts under various conditions facilitated finding conditions under which effective separation of two difficult-to-remove byproducts was achieved by anion exchange (AEX) chromatography. In the current work, as a follow-up study, we demonstrated that the same approach enabled identification of conditions allowing equally good byproduct removal by mixed-mode chromatography with remarkably improved yield. Results from the current and previous studies proved that separation factor determination based on Kp screening for product and byproduct is an effective approach for finding conditions enabling efficient and maximum byproduct removal, especially in challenging cases.

General strategies for IgG-like bispecific antibody purification.

Bispecific antibodies (bsAbs) can simultaneously bind two different antigens or epitopes. Their dual-targeting capability enables novel mechanisms of action, gaining therapeutic advantages over conventional monospecific mAbs. In recent years, the number of bsAbs grows rapidly and bsAbs under development are available in diverse formats. In particular, Fc-containing IgG-like bsAbs, which represent the major group, can be constructed in asymmetric or symmetric format. For asymmetric ones, whose assembly requires multiple distinct chains, although numerous strategies have been developed to promote desired chain pairing, product-related variants such as free chains, half molecules and mispaired species are usually present at various levels. For symmetric ones, increased level of aggregates and truncating variants is often associated with their production. In general, bsAbs pose greater challenges to the downstream team than regular mAbs. In the past few years, our team successfully developed the downstream process for over 70 bsAbs in greater than 30 different formats and accumulated substantial experience. This review introduces general strategies that we have used while purifying these challenging molecules.

Method of understanding for investigation of crack propagation trajectory and fracture aspects in dental cracks on view of fracture mechanics theories.

Current research introduces understanding of dental-cracks mechanistic with fundamental fracture behavior in natural-teeth and orthodontics including mode I crack under both tension and compression, mode II crack under both clockwise-shear and anticlockwise-shear and mixed-mode cracks under both compression-shear and tension-shear. It depends on experimental models of transparent-Plexiglas including pre-cracks of different orientations angle (b) based on fundamental theoretical fracture analysis with comparison. Problem-concept, cracking aspects of fracture-initiation, propagation-direction, fracture-increment length, critical external-load and fracture path are predicted experimentally and theoretically using directional fracture approach and directional strain-energy density theory. Tests are carried out for (36) samples for compression and tension in LEFM. Friction-resistance between crack-surfaces is considered with derivation of equations and charts. Negative stress-intensity factor (-KI) is developed for solving complicated problems of cracks under occlusal compression loads. The occlusal loads are compression and shear producing lateral tensile mixed mode cracks. The critical propagation angle (qc), critical propagation load (sc) and critical propagation envelope of stress intensity factors (KI-KII) are developed with respect to crack orientation angle (b) with comparisons. They are necessary to predict the fracture propagation early before teeth-failure. It helps for prevention and control of dental-cracks, correct-restoration, prosthodontics, orthodontics, and development of new dental-materials and technologies.

Wide-pore fully porous mixed-mode octyl/pyridyl-bonded silica material with pH-dependent surface charge reversal for high-performance hydrophobic charge-induction chromatography of proteins.

In an attempt to overcome silanophilic interactions like observed on popular reversed-phase butyl­bonded silica stationary phases in protein HPLC, a mixed-mode stationary phase based on wide pore silica (3 µm, 300 Å) was prepared by co-immobilization of octyl and 2-pyridylethyl ligands. The surface modification was performed by a new approach using synthesized functional silatranes of the above ligands and prewetted silica. It allowed to generate a dense polymeric siloxane layer on the silica surface. Butyl-bonded silica and octyl/3-aminopropyl-bonded mixed-mode silica phases were prepared for comparison. The modified silicas were subsequently characterized by elemental analysis regarding ligand densities, by solid-state 29Si and 13C cross polarization/magic angle spinning nuclear magnetic resonance spectroscopy for confirming the surface-bonded structure, and by pH-dependent ζ-potential measurements via electrophoretic light scattering providing net surface charge information at distinct pH values. While the classical butyl­bonded stationary phase revealed negative ζ-potential over the entire pH range investigated (pH 3.5-9.5) due to residual silanols and the mixed-mode octyl/3-aminopropyl-bonded silica positive ζ-potential over the entire pH range, pH-dependent charge reversal was observed at approximately pH 5.5 for the octyl/pyridyl-bonded stationary phase. Then, a test set of proteins differing in hydrophobicities and isoelectric points was employed to evaluate the retention characteristics of all three synthesized stationary phases over the pH range of 3 to 7.5 by acetonitrile-gradient elution reversed-phase HPLC. Under acidic conditions (pH 3) the mixed-mode phases octyl/pyridyl-silica and octyl/aminopropyl-silica showed reduced retention and improved peak shapes due to repulsive interactions preventing silanophilic interactions, while protein separations by their hydrophobicities were achieved (repulsive charge-assisted protein RPLC). Finally, the prepared novel mixed-mode octyl/pyridyl-bonded stationary phase was evaluated in hydrophobic charge induction chromatography mode for protein separation of the same test set. Instead of an organic modifier gradient, elution was enforced by a pH gradient from almost neutral to acidic pH at constant organic modifier content of 10 %. This chromatographic mode showed orthogonal retention characteristics and reversed elution order compared to above organic gradient RP-HPLC. In addition, significantly less organic solvent was used under these conditions, classifying it as a green protein LC technology.

Preparation of ionic gel-modified stationary phase for RPLC/HILIC/IC separation and its application in per aqueous liquid chromatography.

In this study, we synthesized and employed an ionic gel-functionalized silica stationary phase for high-performance liquid chromatography. The successful fabrication of the stationary phase was confirmed through attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), zeta-potential measurements, and elemental analysis (EA). Comparative performance evaluation against a commercial column demonstrated the prepared column's effectiveness in the mixed mode of reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), and ion chromatography (IC). Moreover, the stationary phase exhibited exceptional retention repeatability in per aqueous liquid chromatography, showcasing its potential as an environmentally friendly analytical method. Mechanistic investigations unveiled multiple solute-stationary phase interactions, including π-π interactions, hydrogen bonding, and ion exchange. Finally, we applied the developed stationary phase for the precise detection of preservatives in carbonated beverages and jelly, achieving high levels of accuracy and recovery rates.

Dialdehyde cellulose-based mixed-mode stationary phases for diversified high-performance liquid chromatography separations.

To deal with complicated separation situations, this study successfully prepared two mixed-mode chromatography (MMC) stationary phases, CCL-SIL and PCL-SIL, by functionalizing dialdehyde cellulose (DAC) derivatives. In liquid chromatography applications, CCL-SIL exhibited superior separation performance for nucleosides and bases in HILIC mode, while PCL-SIL performed better in RPLC and IEC modes. Their distinct separation mechanisms were also elucidated by quantum chemical calculations. Both CCL-SIL and PCL-SIL showed good stability and reproducibility, with relative standard deviations of retention time, peak area, and peak height below 7.79 % and 4.37 % for multiple injections. Particularly, the PCL-SIL column and the CCL-SIL column were successfully used for the quantitative analysis of trace targets in real samples with complex matrix, demonstrating high accuracy and precision.

Numerical Fatigue Crack Growth on Compact Tension Specimens under Mode I and Mixed-Mode (I+II) Loading.

This study focused on standard Compact Tension (CT) specimens and two loading modes during the numerical analyses carried out, namely: pure mode I and mixed-mode loading (Modes I+II). Numerical stress intensity factors, KI, were calculated using Abaqus® 2022 and compared with those given analytically under pure mode I loading, showing very good agreement. Additionally, KI, KII, and KIII results obtained from Abaqus® were presented for mixed-mode loading, analyzing crack growth and variation through the thickness of the CT specimen. Moreover, fatigue crack growth simulations under mode I loading were conducted on standard CT specimens using the Extended Finite Element Method (XFEM) and the Paris Law parameters of an AISI 316L stainless steel. It was shown that XFEM effectively determines crack propagation direction and growth, provided that an appropriate mesh is implemented.

Recruiting people with selected citizenships for the health interview survey GEDA Fokus throughout Germany: evaluation of recruitment efforts and recommendations for future research.

BACKGROUND: Germany is the second most common country of immigration after the US. However, people with own or familial history of migration are not represented proportionately to the population within public health monitoring and reporting. To bridge this data gap and enable differentiated analyses on migration and health, we conducted the health interview survey GEDA Fokus among adults with Croatian, Italian, Polish, Syrian, or Turkish citizenship living throughout Germany. The aim of this paper is to evaluate the effects of recruitment efforts regarding participation and sample composition. METHODS: Data collection for this cross-sectional and multilingual survey took place between 11/2021 and 5/2022 utilizing a sequential mixed-mode design, including self-administered web- and paper-based questionnaires as well as face-to-face and telephone interviews. The gross sample (n = 33436; age range 18-79 years) was randomly drawn from the residents' registers in 120 primary sampling units based on citizenship. Outcome rates according to the American Association for Public Opinion Research, the sample composition throughout the multistage recruitment process, utilization of survey modes, and questionnaire languages are presented. RESULTS: Overall, 6038 persons participated, which corresponded to a response rate of 18.4% (range: 13.8% for Turkish citizenship to 23.9% for Syrian citizenship). Home visits accounted for the largest single increase in response. During recruitment, more female, older, as well as participants with lower levels of education and income took part in the survey. People with physical health problems and less favourable health behaviour more often took part in the survey at a later stage, while participants with symptoms of depression or anxiety more often participated early. Utilization of survey modes and questionnaire languages differed by sociodemographic and migration-related characteristics, e.g. participants aged 50 years and above more often used paper- than web-based questionnaires and those with a shorter duration of residence more often used a translated questionnaire. CONCLUSION: Multiple contact attempts, including home visits and different survey languages, as well as offering different modes of survey administration, increased response rates and most likely reduced non-response bias. In order to adequately represent and include the diversifying population in public health monitoring, national public health institutes should tailor survey designs to meet the needs of different population groups considered hard to survey to enable their survey participation.

Mixed-Mode Adsorption of l-Tryptophan on D301 Resin through Hydrophobic Interaction/Ion Exchange/Ion Exclusion: Equilibrium and Kinetics Study.

The adsorption of l-tryptophan (l-Trp) was studied based on the hydrophobic interaction/ion exchange/ion exclusion mixed-mode adsorption resin D301. Firstly, the interaction mode between l-Trp and resin was analyzed by studying the influence of pH variation on the adsorption capability and the dissociation state of l-Trp. Secondly, the adsorption mechanism was illuminated by studying the adsorption equilibrium and kinetic behaviors. The adsorption equilibrium and a kinetics model were constructed. The augmentation of pH gradually elicited an enhancement in the adsorption capacity of l-Trp. l-Trp existing in varied dissociation states could be adsorbed by the resin, and the interaction mode relied upon the pH of the solution. An integrated adsorption equilibrium model with the coadsorption of different dissociation states of l-Trp was developed and could predict the adsorption isotherms at various pH levels satisfactorily. Both external mass transfer and intra-particle diffusion collectively imposed constraints on the mass transfer process of l-Trp onto the resin. An improved liquid film linear driving force model (ILM) was constructed, and the model provided a satisfactory fit for the adsorption kinetics curves of l-Trp at various pH levels. l-Trp molecules had a high mass transfer rate at a relatively low solution pH.

Physics-Based Artificial Neural Network Assisting in Extracting Transient Properties of Extrinsically Triggering Photoconductive Semiconductor Switches.

In this paper, a physics-based ANN assisting method for extracting transient properties of extrinsically triggering photoconductive semiconductor switches (ET-PCSSs) is proposed. It exploits the nonlinear mapping of ANN between transient current (input) and doping concentration (output). According to the basic laws of photoelectric device operating, two types of ANN models are constructed by gaussian and polynomial fitting. The mean absolute error (MAE) of forecasting transient photocurrent can be less than 10 A under low triggering optical powers, which verifies the feasibility of ANN assisting TCAD applied to PCSSs. The results are comparable to computation by Mixed-Mode simulation, yet even thousands of seconds of CPU runtime cost are saved in every period. To improve the robustness of the Poly-ANN predictor, Bayesian optimization (BO) is implemented for minimizing the curl deviation of photocurrent-time curves.

Preparation of dicationic ionic liquid modified silica stationary phase for mixed-mode liquid chromatography and its application for food additive detection.

BACKGROUND: Food safety has become an essential aspect of public concern and there are lots of detection means. Liquid chromatography plays a dominating role in food safety inspection because of its high separation efficiency and reproducibility. However, with the increasing complexity of real samples and monitoring requirements, conventional single-mode chromatography would require frequent column replacement and cannot separate different kinds of analytes on a single column simultaneously, which is costly and time-consuming. There is a great need for fabricating mixed-mode stationary phases and validating the feasibility of employing mixed-mode stationary phases for food safety inspection. RESULTS: This work fabricated multifunctional stationary phases for liquid chromatography to determine diverse food additives under the mixed mode of RPLC/HILIC/IEC. Two dicationic ionic liquid silanes were synthesized and bonded onto the silica gel surface. The functionalized silica was characterized by Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and elemental analysis. Both columns provide satisfactory separation performance towards 6 hydrophilic nucleosides, 4 hydrophobic polycyclic aromatic hydrocarbons, and 5 anions. Great repeatability of retention (RSD <0.1 %) and column efficiency (100330 plate/m) were obtained. Thermomechanical analysis and linear solvation energy relationship investigated the retention mechanism. Finally, the better in two prepared columns was employed to separate and determine the contents of NO2- and NO3- in vegetables(highest 4906 mg kg-1 NO3- in spinach), preservatives in bottled beverages (180.8 mg kg-1 sodium benzoate in soft drink), and melamine in milk with satisfactory performance and recovery rates ranging from 96.4 % to 105.6 %. SIGNIFICANCE: This work developed a novel scheme for preparing mixed-mode stationary phases by dicationic ionic liquid which provides great separation selectivity. Most importantly, this work proved the superiority of employing mixed-mode stationary phases for food safety inspection, which might avoid high-cost and frequent changes of columns and chromatography systems in the near future.

Characterization of a mixed mode fluorocarbon/weak anion exchange sorbent for the separation of perfluoroalkyl substances.

The ubiquitous presence and persistence of per- and polyfluoroalkyl substances (PFAS) in the environment have raised concerns in the scientific community. Current research efforts are prioritizing effective PFAS remediation through novel sorbents with orthogonal interaction mechanisms. Recognized sorption mechanisms between PFAS and sorbents include hydrophobic, electrostatic, and fluorine-fluorine interaction. The interplay of these mechanisms contributes significantly to improved sorption capacity and selectivity in PFAS separations. In this study, a primary/secondary amine-functionalized polystyrene-divinylbenzene (Sepra-WAX) polymer was modified to create a fluorinated WAX resin (Sepra-WAX-KelF-PEI). The synthesis intermediate (Sepra-WAX-KelF) was also tested to assess the improvement of the final product (Sepra-WAX-KelF-PEI). The adsorption capacity of Sepra-WAX, Sepra-WAX-KelF, and Sepra-WAX-KelF-PEI, and their interactions with PFAS were evaluated. The effect of pH, ionic strength, and organic solvents on PFAS sorption in aqueous solution was also investigated. The sorbents showed varied adsorption capacities for perfluorooctanoic acid, perfluoropentanoic acid, perfluoro-n-decanoic acid, and hexafluoropropylene oxide dimer acid, with the average extraction capacity of the four analytes being Sepra-WAX-KelF-PEI (523 mg/g) > Sepra-WAX (353 mg/g) > Sepra-WAX-KelF (220 mg/g). Sepra-WAX-KelF-PEI provided the highest adsorption capacity for all analytes tested, proving that the combination of electrostatic and hydrophobic/fluorophilic interactions is crucial for the effective preconcentration of PFAS and its future applications for PFAS remediation from aqueous solutions.

Automated Liquid Handling Extraction and Rapid Quantification of Underivatized Amino Acids and Tryptophan Metabolites from Human Serum and Plasma Using Dual-Column U(H)PLC-MRM-MS and Its Application to Prostate Cancer Study.

Amino acids (AAs) and their metabolites are important building blocks, energy sources, and signaling molecules associated with various pathological phenotypes. The quantification of AA and tryptophan (TRP) metabolites in human serum and plasma is therefore of great diagnostic interest. Therefore, robust, reproducible sample extraction and processing workflows as well as rapid, sensitive absolute quantification are required to identify candidate biomarkers and to improve screening methods. We developed a validated semi-automated robotic liquid extraction and processing workflow and a rapid method for absolute quantification of 20 free, underivatized AAs and six TRP metabolites using dual-column U(H)PLC-MRM-MS. The extraction and sample preparation workflow in a 96-well plate was optimized for robust, reproducible high sample throughput allowing for transfer of samples to the U(H)PLC autosampler directly without additional cleanup steps. The U(H)PLC-MRM-MS method, using a mixed-mode reversed-phase anion exchange column with formic acid and a high-strength silica reversed-phase column with difluoro-acetic acid as mobile phase additive, provided absolute quantification with nanomolar lower limits of quantification within 7.9 min. The semi-automated extraction workflow and dual-column U(H)PLC-MRM-MS method was applied to a human prostate cancer study and was shown to discriminate between treatment regimens and to identify metabolites responsible for discriminating between healthy controls and patients on active surveillance.

Ionic-liquid/Carbowax 20 M functionalized capsule phase microextraction platform for the extraction of phosphodiesterase-5 inhibitors from human serum and urine prior to their determination by LC-MS.

Capsule phase microextraction (CPME) is an efficient bioanalytical technique that streamlines the sample preparation by integrating the filtration and stirring mechanism directly into the device. A novel composite sorbent designed to be selective towards the target analytes consisting of mixed-mode sorbent chemistry synthesized by sol-gel technology is found promising and superior to the conventional C18 sorbents. Herein we describe the encapsulation of an ionic liquid (IL)/Carbowax 20M-functionalized sol-gel sorbent (sol-gel IL/Carbowax 20 M) in the lumen of porous polypropylene tubes for the capsule phase microextraction of three phosphodiesterase-5 inhibitors namely avanafil, sildenafil, and tadalafil in human serum and urine samples. The CPME device was characterized by Scanning Electron Microscopy (SEM) and Fourier-Transform Infrared Spectroscopy (FT-IR). The experimental parameters of CPME procedure (e.g. sample pH and ionic strength, extraction time, stirring rate, elution solvent and volume) were carefully optimized to achieve the highest possible extraction efficiency for the analytes. Method validation was conducted in terms of precision, linearity, accuracy, matrix effect, lower limits of quantification, and limits of detection (LOD). The method linearity was investigated in the range of 50-1000 ng mL-1 for all analytes while the precision was less than 11.8 % in all cases. For all analytes, the LOD values were 17 ng mL-1. The IL/CW 20M-functionalized microextraction capsules could be reused at least 25 times both for urine and serum samples. The green character and the applicability of the proposed method were evaluated using the ComplexGAPI and BAGI indexes. The optimized CPME protocol exhibited reduced consumption of organic solvent and generation of waste, cost-effectiveness, and simplicity. Finally, the proposed method was successfully applied to the analysis of sildenafil in human urine after administration of drug-containing formulation.

Monitoring of the disulfide scrambled species by mixed-mode SEC-HPLC during the purification of a bispecific antibody.

Size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) is an analytical method routinely used for assessing aggregation content in protein samples. As SEC-HPLC separates analytes based on their hydrodynamic radius, it generally lacks the capability of differentiating species that are similar in size. Recently while purifying a bispecific antibody (bsAb), we noticed that SEC-HPLC can provide certain degree of resolution between the target bsAb and a disulfide scrambled form, although these two species were identical in molecular weight. In seeing the unexpected potential of SEC-HPLC at resolving species with similar size, we further tested Zenix SEC-300, a mixed-mode SEC-HPLC column from Sepax, which was reported to be capable of separating protein analytes based on other factors besides size. The Zenix column indeed provided resolution much better than the regular SEC-HPLC column. Upon further optimization, the Zenix column allowed close to baseline separation of the correctly folded and the disulfide scrambled species. The current study, as a complement to the previous reports, further demonstrates that mix-mode SEC-HPLC is capable of separating protein analytes that are close in size but are different in conformation and/or surface characteristics.

Novel PEI/Zein core-shell composite as mixed-mode stationary phase for high performance liquid chromatography.

Based on the adhesion of polyethyleneimine (PEI), a novel PEI/zein co-modified core-shell stationary phase (PEI/Zein@SiO2) was prepared by doping zein to form a composite modification layer. The stationary phase achieved effective separation of nucleosides, bases and antibiotics in hydrophilic interaction mode on account of the hydrophilic groups of composite coating. With the hydrophobicity of zein, the flavones could be separated in reversed-phase mode. In short, the separation and analysis of hydrophilic/hydrophobic compounds were accomplished excellently by the PEI/Zein@SiO2 column with mixed double mode. The prepared chromatographic stationary phase not only avoided the dissolution of zein, but also covered the strong adsorption of some analytes caused by silica hydroxyl groups on the surface of silica spheres. The morphological structure and specific surface area of the material were reflected by various characterization techniques. Hydrophilic/hydrophobic compounds were used as tested analytes to research separation performance and retention mechanisms of PEI/Zein@SiO2 column. The stability and reproducibility of the PEI/Zein@SiO2 stationary phase were satisfied. Therefore, the modification of zein could improve the separation selectivity of stationary phase effectively for complex samples, which had the potential to be one of the significant potential application materials in stationary phase packing.

[Recent progress of chromatographic techniques for antibody purification].

Antibody drugs are becoming increasingly popular in disease diagnosis, targeted therapy, and immunoprevention owing to their characteristics of high targeting ability, strong specificity, low toxicity, and mild side effects. The demand for antibody drugs is steadily increasing, and their production scale is expanding. Upstream cell culture technology has been greatly improved by the high-capacity production of monoclonal antibodies. However, the downstream purification of antibodies presents a bottleneck in the production process. Moreover, the purification cost of antibodies is extremely high, accounting for approximately 50%-80% of the total cost of antibody production. Chromatographic technology, given its selectivity and high separation efficiency, is the main method for antibody purification. This process usually involves three stages: antibody capture, intermediate purification, and polishing. Different chromatographic techniques, such as affinity chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, mixed-mode chromatography, and temperature-responsive chromatography, are used in each stage. Affinity chromatography, mainly protein A affinity chromatography, is applied for the selective capture and purification of antibodies from raw biofluids or harvested cell culture supernatants. Other chromatographic techniques, such as ion-exchange chromatography, hydrophobic interaction chromatography, and mixed-mode chromatography, are used for intermediate purification and antibody polishing. Affinity biomimetic chromatography and hydrophobic charge-induction chromatography can produce antibodies with purities comparable with those obtained through protein A chromatography, by employing artificial chemical/short peptide ligands with good selectivity, high stability, and low cost. Temperature-responsive chromatography is a promising technique for the separation and purification of antibodies. In this technique, antibody capture and elution is controlled by simply adjusting the column temperature, which greatly eliminates the risk of antibody aggregation and inactivation under acidic elution conditions. The combination of different chromatographic methods to improve separation selectivity and achieve effective elution under mild conditions is another useful strategy to enhance the yield and quality of antibodies. This review provides an overview of recent advances in the field of antibody purification using chromatography and discusses future developments in this technology.

Analytical QbD for the optimization of a multimode HPLC method for the investigation of hydrochlorothiazide, diltiazem and propranolol release from 3D printed formulation.

Since 3D printing technology is an emerging field in pharmaceutical technology, the present study aimed at the development of a mixed-mode liquid chromatographic method for the separation and determination of hydrochlorothiazide, diltiazem, and propranolol to investigate their in-vitro release performance from 3D printed tablets. Due to the unique properties of the mixed-mode stationary phase, the three drugs were separated in less than 8 min under isocratic elution. Method development was accomplished following the Analytical Quality by Design principles and was evaluated using risk assessment and multivariate analysis. The influences of critical method parameters on critical method attributes (were screened using a 2-level fractional factorial design and subsequently optimized through a central composite design. The method operable design region was approved by the establishment of a robust zone using Monte Carlo simulation and capability analysis. The validation of the HPLC method was performed based on the total error concept. The relative bias was varied between ─ 11.6 % and 10.5 % and the RSD values for repeatability and intermediate precision were below 4.4 % in all cases. The limits of detection (LOD) ranged between 0.17 - 0.90 µg/mL and were adequate for the specific application. The developed method was successfully applied to the analysis of the studied drugs in in-vitro drug release samples obtained from 3D-printed tablets combining the above-mentioned active pharmaceutical ingredients (APIs).

Modelling the pH dependent retention and competitive adsorption of charged and ionizable solutes in mixed-mode and reversed-phase liquid chromatography.

This study investigated the influence of pH on the retention of solutes using a mixed-mode column with carboxyl (-COOH) groups acting as weak cation exchanger bonded to the terminal of C18 ligands (C18-WCX column) and a traditional reversed-phase C18 column. First, a model based on electrostatic theory was derived and successfully used to predict the retention of charged solutes (charged, and ionizable) as a function of mobile phase pH on a C18-WCX column. While the Horváth model predicts the pH-dependent retention of ionizable solutes in reversed-phase liquid chromatography (RPLC) solely based on solute ionization, the developed model incorporates the concept of surface potential generated on the surface of the stationary phase and its variation with pH. To comprehensively understand the adsorption process, adsorption isotherms for these solutes were individually acquired on the C18-WCX and reversed-phase C18 columns. The adsorption isotherms followed the Langmuir model for the uncharged solute and the electrostatically modified Langmuir model for charged solutes. The elution profiles for the single components were calculated from these isotherms using the equilibrium dispersion column model and were found to be in close agreement with the experimental elution profiles. To enable modelling of two-component cases involving charged solute(s), a competitive adsorption isotherm model based on electrostatic theory was derived. This model was later successfully used to calculate the elution profiles of two components for scenarios involving (a) a C18 Column: two charged solutes, (b) a C18 Column: one charged and one uncharged solute, and (c) a C18-WCX Column: two charged solutes. The strong alignment between the experimental and calculated elution profiles in all three scenarios validated the developed competitive adsorption model.

Incorporating Adaptive Survey Design in a Two-Stage National Web or Mail Mixed-Mode Survey: An Experiment in the American Family Health Study.

This article presents the results of an adaptive design experiment in the recruitment of households and individuals for a two-stage national probability web or mail mixed-mode survey, the American Family Health Study (AFHS). In the screening stage, we based the adaptive design's subgroup differentiation on Esri Tapestry segmentation. We used tailored invitation materials for a subsample where a high proportion of the population was Hispanic and added a paper questionnaire to the initial mailing for a subsample with rural and older families. In the main-survey stage, the adaptive design targeted the households where a member other than the screening respondent was selected for the survey. The adaptations included emailing and/or texting, an additional prepaid incentive, and seeking screening respondents' help to remind the selected individuals. The main research questions are (i) whether the adaptive design improved survey production outcomes and (ii) whether combining adaptive design and postsurvey weighting adjustments improved survey estimates compared to performing postsurvey adjustments alone. Unfortunately, the adaptive designs did not improve the survey production outcomes. We found that the weighted AFHS estimates closely resemble those of a benchmark national face-to-face survey, the National Survey of Family Growth, although the adaptive design did not additionally change survey estimates beyond the weighting adjustments. Nonetheless, our experiment yields useful insights about the implementation of adaptive design in a self-administered mail-recruit web or mail survey. We were able to identify subgroups with potentially lower response rates and distinctive characteristics, but it was challenging to develop effective protocol adaptations for these subgroups under the constraints of the two primary survey modes and the operational budget of the AFHS. In addition, for self-administered within-household selection, it was difficult to obtain contact information from, reach, and recruit selected household members that did not respond to the screening interview.