Intermolecular Interactions, Solute Descriptors, and Partition Properties of Neutral Per- and Polyfluoroalkyl Substances (PFAS).

The environmental partition properties of perfluoroalkyl and polyfluoroalkyl substances (PFAS) must be understood for their transport and fate analysis. In this study, isothermal gas chromatographic (GC) retention times of 60 neutral PFAS were measured using four columns with different stationary phase polarities, which indicated varying polar interactions exerted by these substances. The GC data were combined with new octanol/water partition coefficient data from this study and existing partition coefficient data from the literature and used to determine the polyparameter linear free energy relationship (PP-LFER) solute descriptors. A complete set of the solute descriptors was obtained for 47 PFAS, demonstrating the characteristic intermolecular interaction properties, such as hydrogen bonding capabilities influenced by the electron-withdrawing perfluoroalkyl group. The partition coefficients between octanol and water, air and water, and octanol and air predicted by the PP-LFER models agreed with those predicted by the quantum chemically based model COSMOtherm, suggesting that both models are highly accurate for neutral PFAS and can fill the current large data gaps in partition property data. A chemical partitioning space plot was generated by using the PP-LFER-predicted partition coefficients, showing the primary importance of the air phase for the environmental distribution of nonpolar and weakly polar PFAS and the increasing significance of organic phases with increasing PFAS polarity.

Revisiting the dependence of retention factor on the content of organic component in the mobile phase in reversed-phase HPLC.

In high-performance liquid chromatography, the dependence of retention factor k on volumetric fraction ϕ of organic phase is expressed by log k = F(ϕ) with F(ϕ) obtained by measuring log k at different ϕ values. From F(ϕ), a value kw is calculated by taking ϕ = 0. The equation log k = F(ϕ) is applied for predicting k, and kw is a descriptor of hydrophobic character of solutes and stationary phases. Calculated kw should not depend on the nature of organic component of mobile phase but extrapolation procedure leads to different kw for different organic components. The present study shows that the expression of F(ϕ) changes depending on the range of ϕ and the same function F(ϕ) cannot be used for the full range of ϕ from 0 to 1. Consequently, kw obtained by extrapolation of ϕ to zero is not correct because the expression of F(ϕ) was generated by fitting the data using ϕ with higher values. The present study shows the proper way to obtain the value of kw .

Application of Ionic Liquids as Mobile Phase Additives for Simultaneous Analysis of Nicotine and Its Metabolite Cotinine in Human Plasma by HPLC-DAD.

Nicotine and cotinine are very polar basic molecules, which makes it difficult to analyze them by reversed-phase liquid chromatography (RPLC), especially in biological samples. Additives with an ionic character have been traditionally used in RPLC as silanol suppressors. The aim of our study was to investigate the potential of selected ionic liquids in improving chromatographic performance in comparison with common additives. The experimental design was conducted using the following ionic liquids as the mobile phase modifiers: 1-butyl-3-methylimidazolium tetrafluoroborate, BMIM[BF4] and 1-butyl-3-methylimidazolium hexafluorophosphate BMIM[PF6], with a C18 chromatographic column. The separation of these alkaloids on silica-based RPLC stationary phases was successfully conducted by the addition of BMIM[BF4] in an acetonitrile:phosphate-buffer-based mobile phase in a pH range of 2.3-5.2. The presented chromatographic method can be used as alternative for monitoring studies or pharmacokinetic application necessary for the evaluation of tobacco smoke exposure.

Accuracy of retention model parameters obtained from retention data in liquid chromatography.

In liquid chromatography, it is often very useful to have an accurate model of the retention factor, k, over a wide range of isocratic elution conditions. In principle, the parameters of a retention model can be obtained by fitting either isocratic or gradient retention factor data. However, in spite of many of our own attempts to accurately predict isocratic k values using retention models trained with gradient retention data, this has not worked in our hands. In the present study, we have used synthetic isocratic and gradient retention data for small molecules under reversed-phase liquid chromatography conditions. This allows us to discover challenges associated with predicting isocratic k values without the confounding influences of experimental issues that are difficult to model or eliminate. The results indicate that it is not currently possible to consistently predict isocratic retention factors for small molecules with accuracies better than 10%, even when using synthetic gradient retention data. Two distinct challenges in fitting gradient retention data were identified: 1) a lack of 'uniqueness' in the parameters and 2) an inability to find the global optimum fit in a complex fitting landscape. Working with experimental data where measurement noise is unavoidable will only make the accuracy worse.

Lipophilicity as a Central Component of Drug-Like Properties of Chalchones and Flavonoid Derivatives.

Lipophilcity is an important physico-chemical parameter that influences membrane transport and binding ability to action. Migration distance following complete elution of compounds was used to calculate different lipophilicity-related parameters. The aim of this study is to show that lipophilicity is a central component of thiazole chalcones and flavonoid derivatives regarding their drug-like properties. Experimental and computational methods were used. This study considers 44 previously synthesized compounds (thiazole chalcones, flavanones, flavones, 3-hydroxyflavones, and their acetylated derivatives). The concerned compounds have shown antitumoral hallmarks and antibacterial activity in vitro. The experimental method used to determine compounds' lipophilicity was the reverse-phase thin layer chromatography (RP-TLC). Lipophilicity related parameters-isocratic retention factor (RM), relative lipophily (RM0), slope (b), chromatographic hydrophobic index (φ0), scores of principal components (PC1/RM)-were determined based on reverse-phase chromatography results.

Evaluation of affinity interaction between small molecules and platelets by open tubular affinity capillary electrochromatography.

In this paper, an open tubular affinity capillary electrochromatography (OT-ACEC) was developed by physical adsorption of rabbit platelets on the inner surface of capillary. The interactions between small molecules include adenosine diphosphate (ADP) (positive control), protocatechuic acid (negative control) and seven natural products (salvianolic acid B, salvianic acid A sodium, hydroxysafflor yellow A, ferulic acid, chlorogenic acid, sinapic acid, caffeic acid) and platelets were evaluated by their retention factors and binding constants obtained based on peak-shift assay. Then, the activities of anti-platelet aggregation induced by thrombin (THR), ADP and arachidonic acid (AA) for those small molecules (except ADP) were evaluated by turbidimetric method. The results indicate that: (i) ADP, a platelet aggregation inducer, had strong interaction with platelet, while protocatechuic acid that had no inhibition on platelet aggregation behaved no specific interaction; (ii) there was a positive correlation between the anti-platelet aggregation activities of small molecules and their interactions with platelet, generally those compounds with higher binding constants with platelet exhibited higher activities. Therefore, the OT-ACEC method developed in the present study can be a potential method to evaluate affinity interactions between small molecules and platelets, so as to predict the biological activities such as anti-platelet aggregation for the small molecules.

Prediction of retention characteristics of heterocyclic compounds.

The CORAL software ( http://www.insilico.eu/coral ) was used to build up quantitative structure-property relationships (QSPRs) for the retention characteristics of 93 derivatives of three groups of heterocyclic compounds: 2-phenyl-1,3-benzoxazoles, 4-benzylsulfanylpyridines, and benzoxazines. The QSPRs are one-variable models based on the optimal descriptors calculated from the molecular structure represented by simplified molecular input-line entry systems (SMILES). Each symbol (or two undivided symbols) of SMILES is characterized by correlation weight. The optimal descriptor is the sum of the correlation weights. The numerical data on the correlation weights were calculated with the Monte Carlo method by the manner which provides best correlation between endpoint and optimal descriptor for the calibration set. The predictive ability of the model is checked with the validation set (compounds invisible during building up of the model). The approach has been checked with three random splits into the training, calibration, and validation sets: all models have apparent predictive potential. The mechanistic interpretation of the molecular features extracted from SMILES as the promoters of increase or decrease of examined endpoints is suggested.

The BRCA1-binding protein BRAP2 can act as a cytoplasmic retention factor for nuclear and nuclear envelope-localizing testicular proteins.

Regulation of nuclear protein import is central to many cellular processes such as development, with a key mechanism being factors that retain cargoes in the cytoplasm that normally localize in the nucleus. The breast cancer antigen BRCA1-binding protein BRAP2 has been reported as a novel negative regulator of nuclear import of various nuclear localization signal (NLS)-containing viral and cellular proteins, but although implicated in differentiation pathways and highly expressed in tissues including testis, the gamut of targets for BRAP2 action in a developmental context is unknown. As a first step towards defining the BRAP2 interactome, we performed a yeast-2-hybrid screen to identify binding partners of BRAP2 in human testis. Here we report characterization for the first time of three of these: the high mobility group (HMG)-box-domain-containing chromatin component HMG20A, nuclear mitotic apparatus protein NuMA1 and synaptic nuclear envelope protein SYNE2. Co-immunoprecipitation experiments indicate association of BRAP2 with HMG20A, NuMA1, and SYNE2 in testis, underlining the physiological relevance of the interactions, with immunohistochemistry showing that where BRAP2 is co-expressed with HMG20A and NuMA1, both are present in the cytoplasm, in contrast to their nuclear localization in other testicular cell types. Importantly, quantitative confocal microscopic analysis of cultured cells indicates that ectopic expression of BRAP2 inhibits nuclear localization of HMG20A and NuMA1, and prevents nuclear envelope accumulation of SYNE2, the first report of BRAP2 altering localization of a non-nuclear protein. These results imply for the first time that BRAP2 may have an important role in modulating subcellular localization during testicular development.

Processes involved in sweeping as sample enrichment method in cyclodextrin-modified micellar electrokinetic chromatography of hydrophobic basic analytes.

Sweeping is an enrichment method in MEKC, which includes following steps: stacking/destacking of the micelles, sweeping of analyte by the stacked/destacked micelles, destacking/stacking of the swept analyte zone and additional focusing/defocusing due to the retention factor gradient effect (RFGE). In this study, we investigate additional processes, regarding online focusing in cyclodextrin-modified MEKC (CD-MEKC) of hydrophobic basic analytes: dynamic pH junction (sample with pH different from that of BGE) and adsorption of analyte onto the capillary wall within the sample zone. It is demonstrated that the developed method for the assessment of the sweeping efficiency is also applicable to CD-MEKC taking ethylparaben as an example of acidic analytes and desloratadine as an example of basic analytes using different types of ß-cyclodextrin. Our previous results regarding RFGE as an additional focusing/defocusing effect in sweeping-MEKC are confirmed for the case that the apparent distribution coefficient differs for the sample and the BGE due to a different content of the complex-forming agent cyclodextrin and due to a pH difference between the sample and the BGE. Despite being significantly more hydrophobic than ethylparaben, desloratadine shows an unexpectedly low enrichment factor. This enrichment factor is nearly unaffected by the addition of CD to the BGE. This unexpected behavior is attributed to wall adsorption of the protonated hydrophobic basic analyte within the sample zone, which significantly counteracts the sweeping process. This assumption is corroborated by an improvement in the enrichment factor achieved via addition of a dynamic coating agent (triethylamine) to the sample solution.

A logarithmic law for the velocity- and retention-dependency of the eddy dispersion in chromatographic columns.

Applying the recently introduced patchwork model for porous media, we present a new step forward in the modelling of eddy dispersion in chromatographic columns. The logarithmic law describing the velocity dependency emerging from this patchwork model is supplemented with a retention factor dependency via first principles modelling of the variations in flow resistance and retention capacity caused by the packing disorder. Furthermore, it is shown the derived expression is also able to fit the eddy dispersion originating from the wall effect on the packing. When applied to literature data of eddy dispersion, the newly introduced logarithmic law has a goodness of fit that is at least equal to that of Knox' empirical power law (R2>0.98). The main difference is that, whereas Knox' power law requires a separate fit for each component due to the retention factor dependency, the present model simultaneously fits all plate height curves measured on one chromatographic column, using only two parameters with a clear physical meaning.

Comparison of chiral separations of aminophosphonic acids and their aminocarboxylic acid analogs using a crown ether column.

Crown ethers are capable of complexing with primary amines and have been utilized in chromatography to separate amino acid racemates. This application has been extended to resolve (1-amino-1-phenylmethyl)phosphonic acid and (1-aminoethyl)phosphonic acid racemates, along with their aminocarboxylic acid analogs (2-phenylglycine and alanine, respectively), via a ChiroSil RCA crown ether based chiral stationary phase. Effects of the organic modifier, temperature, and acid type and concentration on retention and selectivity were also investigated. Trends in retention and selectivity varied between aminophosponic acids and their aminocarboxylic analogs. Computer modeling and (1)H NMR analyses were performed to potentially gain a better understanding of interactions of the aforementioned molecules with the ChiroSil RCA chiral stationary phase. Theoretical predictions of the most stable conformations for (R)- and (S)-enantiomers were compared to elution order; it was found that the elution order agreed with molecular modeling such that the longest retention correlated with the predicted most stable complex between the enantiomer and crown ether. (1)H NMR demonstrated interactions of aminophosphonic and aminocarboxylic racemates with (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid in solution and was utilized to determine enantiomeric excess of (1-amino-1-phenylmethyl)phosphonic acid after its enantioenrichment via crystallization through diastereomeric salt formation with the crown ether followed by filtration.

Transport diameters of liquid chromatography columns.

The transport diameter (dM) of a chromatographic column is an objective (measurable) representation of cross-sectional dimensions of the column flow-through channels, and of the effect of porosity of the column internal support structure on velocity of transporting solvent molecules along the column. A column kinetic performance is trade-off between the column separation performance, analysis time and pressure. The dM is a key factor in definition of the kinetic performance factor - an objective metric of a column kinetic performance - and of other objective performance metrics. General equations for evaluation of dM are developed. The dM of PLOT, particulate and pillar-array columns are evaluated and discussed.

Experimental design and re-parameterization of the Neue-Kuss model for accurate and precise prediction of isocratic retention factors from gradient measurements in reversed phase liquid chromatography.

The present work describes a re-parameterization of the Neue Kuss (NK) model for describing retention in liquid chromatography, and this re-parameterized model is used to fit a large set of isocratic retention measurements with improved convergence properties relative to the original parameterization of the model. Next, an experimental design for retention measurements using mobile phase gradient elution conditions is proposed for the purpose of obtaining accurate and precise NK parameters. Simulated retention data for mobile phase gradient elution conditions with two different levels of noise, as well as an essentially zero noise level were fit with the re-parameterized model. The results showed that the re-parameterized fits yielded average (absolute value) prediction errors for the parameters at the highest noise level of 7.2 % for S1,ref, 18 % for S2,ref and 6.2 % for kref (the re-parameterized NK model parameters). These errors were significantly smaller than those for the original parameterization of the NK model, where the errors were 23 % for S1, 25 % for S2 and 160 % for kw (the original NK model parameters). Furthermore, isocratic retention factors predicted using these model parameters were found to have an average magnitude of error of 0.51 % for the re-parameterized model, as opposed to 6800 % for the model with the original parameterization. A further test of this approach was carried out for independent experimental measurements for five solutes on a C18 column. The average magnitude of error of the isocratic retention factors predicted from parameters obtained from fits of gradient data was 1.6 %, provided that the range of organic solvent compositions that the solute sampled in the mobile phase gradient experiments was consistent with the isocratic experiments. These results indicate that the re-parameterization of the NK model allows for significant improvements in the fitting process, and that the proposed experimental design allows for NK parameters to be extracted from mobile phase gradient experiments, with prediction accuracies of isocratic retention factors on the order of 1-2 %.

Effect of time-invariant pressure gradients on peak formation and efficiency in ultrahigh-pressure liquid chromatography.

In chromatography, pressure can affect the retention factors of compounds significantly. In liquid chromatography, this effect is primarily related to the change in the molecular volume of solute during adsorption that is remarkably high for large biomolecules such as peptides and proteins. As a result, the migration velocities of chromatographic bands vary spatially through the column affecting the degree of band broadening. In this work, based on theoretical considerations, chromatographic efficiencies are studied under pressure-induced gradient conditions. The retention factor and migration velocity of different components are examined, and it is shown that components with the same retention time can have different migration patterns. The width of the initial band after injection is affected by the pressure gradient, providing significantly thinner initial bands for compounds with higher pressure sensitivity. In addition to classical band broadening phenomena, the influence of pressure gradients on band broadening is remarkable. The positive velocity gradient leads to extra band broadening. Our results clearly demonstrate that the zones are significantly wider at the end of the column if the change of molar volume of solute during adsorption is large. If the pressure drop is increasing, this effect becomes more significant. In the same time, the high release velocity of the bands somewhat counteracts the extra band broadening effect, however, it can not offset it perfectly. As a result, the separation efficiency of large biomolecules is decreased significantly due to the chromatographic pressure gradient. Under UHPLC conditions, the extent of apparent efficiency loss can reach up to 50% compared to the intrinsic efficiency of the column.

Simultaneous effect of different chromatographic conditions on the chromatographic retention of pentapeptide derivatives (HGRFG and NPNPT).

Introduction: Oligopeptides exhibit great prospects for clinical application and its separation is of great importance in new drug development. Methods: To accurately predict the retention of pentapeptides with analogous structures in chromatography, the retention times of 57 pentapeptide derivatives in seven buffers at three temperatures and four mobile phase compositions were measured via reversed-phase high-performance liquid chromatography. The parameters ( k H A , k A , and p K a ) of the acid-base equilibrium were obtained by fitting the data corresponding to a sigmoidal function. We then studied the dependence of these parameters on the temperature (T), organic modifier composition (φ, methanol volume fraction), and polarity ( P m N parameter). Finally, we proposed two six-parameter models with (1) pH and T and (2) pH and φ or P m N as the independent variables. These models were validated for their prediction capacities by linearly fitting the predicted retention factor k-value and the experimental k-value. Results: The results showed that log k H A and log k A exhibited linear relationships with 1 / T , φ or P m N for all pentapeptides, especially for the acid pentapeptides. In the model of pH and T, the correlation coefficient (R2) of the acid pentapeptides was 0.8603, suggesting a certain prediction capability of chromatographic retention. Moreover, in the model of pH and φ or P m N , the R2 values of the acid and neutral pentapeptides were greater than 0.93, and the average root mean squared error was approximately 0.3, indicating that the k-values could be effectively predicted. Discussion: In summary, the two six-parameter models were appropriate to characterize the chromatographic retention of amphoteric compounds, especially the acid or neutral pentapeptides, and could predict the chromatographic retention of pentapeptide compounds.

The Effect of Flake Production and In Vitro Digestion on Releasing Minerals and Trace Elements from Wheat Flakes: The Extended Study of Dietary Intakes for Individual Life Stage Groups.

This thorough study analyses the amounts of 43 minerals and trace elements in non-traditional wheat grains, flakes, and undigested flake portions using ICP-MS and establishes declines in their respective contents after the flake production. It also identifies appropriate dietary intakes, in vitro digestibility values, retention factors, and metal pollution indexes. The element contents in wheat flakes are lower than in wheat grains after the hydrothermal treatment process, and their declines are: Na (48-72%), Ce (47-72%), Sr (43-55%), Tl (33-43%), Ti (32-41%), U (31-44%), Ho (29-69%), Cr (26-64%), Zr (26-58%), Ag (25-52%), and Ca (25-46%). The flakes significantly contributed to the recommended dietary intake or adequate intake of particular elements for men of all categories as follows: Mn (143%) > Mo > Cu > Mg ≥ Cr > Fe (16%); for women: Mn (up to 183%) > Mo > Cu > Cr ≥ Mg > Fe (7-16%); for pregnant women aged 19-30: Mn (165%) > Mo > Cu > Mg > Cr (25%); and finally, for lactating women: Mn (127%) > Mo > Cu > Mg > Cr (17%). The contributions to the provisional tolerable weekly or monthly intakes of all toxic elements were established as being within the official limits. The daily intakes for non-essential elements were also calculated. The retention factors were calculated to assess the element concentrations in the undigested part using the digestibility values (87.4-90.5%). The highest retention factors were obtained for V (63-92%), Y (57-96%), Ce (43-76%), Pb (34-58%), Tl (32-70%), Ta (31-66%), and Ge (30-49%). K, Mg, P, Zn, Ba, Bi, Ga, Sb, Cu, Ni, and As appear to be released easily from flake matrices during digestion. The metal pollution index has been confirmed as being lower for non-traditional wheat flakes when compared with grains. Importantly, 15-25% of the metal pollution index assessed for native flakes remains in the undigested flake portion after in vitro digestion.

Metabolic reprogramming of hepatocytes by Schistosoma mansoni eggs.

Background & Aims: Schistosomiasis is a parasitic infection which affects more than 200 million people globally. Schistosome eggs, but not the adult worms, are mainly responsible for schistosomiasis-specific morbidity in the liver. It is unclear if S. mansoni eggs consume host metabolites, and how this compromises the host parenchyma. Methods: Metabolic reprogramming was analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging, liquid chromatography with high-resolution mass spectrometry, metabolite quantification, confocal laser scanning microscopy, live cell imaging, quantitative real-time PCR, western blotting, assessment of DNA damage, and immunohistology in hamster models and functional experiments in human cell lines. Major results were validated in human biopsies. Results: The infection with S. mansoni provokes hepatic exhaustion of neutral lipids and glycogen. Furthermore, the distribution of distinct lipid species and the regulation of rate-limiting metabolic enzymes is disrupted in the liver of S. mansoni infected animals. Notably, eggs mobilize, incorporate, and store host lipids, while the associated metabolic reprogramming causes oxidative stress-induced DNA damage in hepatocytes. Administration of reactive oxygen species scavengers ameliorates these deleterious effects. Conclusions: Our findings indicate that S. mansoni eggs completely reprogram lipid and carbohydrate metabolism via soluble factors, which results in oxidative stress-induced cell damage in the host parenchyma. Impact and implications: The authors demonstrate that soluble egg products of the parasite S. mansoni induce hepatocellular reprogramming, causing metabolic exhaustion and a strong redox imbalance. Notably, eggs mobilize, incorporate, and store host lipids, while the metabolic reprogramming causes oxidative stress-induced DNA damage in hepatocytes, independent of the host's immune response. S. mansoni eggs take advantage of the host environment through metabolic reprogramming of hepatocytes and enterocytes. By inducing DNA damage, this neglected tropical disease might promote hepatocellular damage and thus influence international health efforts.

Prediction of surface excess adsorption and retention factors in reversed-phase liquid chromatography from molecular dynamics simulations.

An alternative method to the classical fit of semi-empirical, statistical, or artificial intelligence-based models to retention data is proposed to predict surface excess adsorption and retention factors in liquid chromatography. The approach is based on a fundamental, microscopic description of the liquid-to-solid adsorption of analytes taking place at the interface between a bulk liquid phase and a solid surface. Molecular dynamics (MD) simulations are performed at T=300 K in a 100 Å wide slit-pore model (ß-cristobalite-C18 surface in contact with an acetonitrile/water mobile phase) to quantify a priori the retention factors of small molecules expected in reversed phase liquid chromatography (RPLC). Uracil is chosen as the reference "non-retained" marker, whereas benzyl alcohol, acetophenone, benzene, and ethylbenzene are four selected retained, neutral compounds. The MD simulations allow to determine the pore-level density profiles of these five compounds, i.e., the variation of the analyte concentration as a function of distance from the silica surface. The retention factors of the retained analytes are expressed using their respective calculated surface excess adsorption relative to uracil. By definition, the retention factors are proportional to the surface excess adsorbed and the proportionality constant is directly scaled to the retention time of the "non-retained" marker. Experimentally, a 4.6 mm × 150 mm RPLC-C18 column packed with 5 µm 100 Å High Strength Silica (HSS)-C18 particles is used and the retention times of these five compounds are measured. The volume fraction of acetonitrile in water increases from 20 to 90% generating a wide range of retention factors from 0.15 to 183 at T=300 K. The results demonstrate very good agreement between the MD-predicted surface excess adsorption data and measured retention factors (R2> 0.985). A systematic error is observed as the proportionality constant is not exactly scaled to the retention time of uracil. This is most likely caused by the differences between the chemical and morphological features of the slit-pore model adopted in the MD simulations and those of the actual HSS-C18 particles: the average surface coverage with C18 chains, the geometry of the mesopores, and the pore size distribution. Specifically, the impact on RPLC retention of slight, local variations in surface chemistry (e.g., functional group density and uniformity) and how this aspect is affected by the pore space morphology (e.g., pore curvature and size) is worth investigating by future MD simulations.

Preparation of covalently bonded liposome capillary column and its application in evaluation of drug membrane permeability.

Two liposomes, including 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) + 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (PE) + cholesterol (Chol) (DSPC/PE/Chol liposomes) and soybean lecithin (SPC) + PE + Chol (SPC/PE/Chol liposomes), were prepared and fixed on the inner wall of capillary column by using the adhesion of polydopamine (PDA) membrane and the cross-linking property of glutaraldehyde (GA). The immobilized liposome capillary column (ILCC) has good repeatability and stability based on the electrophoretic mobility of analyte. A CE method based on the immobilized liposome capillary column chromatography (ILCCC) was successfully developed to study the retention behavior of drugs on ILCC, and the logarithm of retention factor (log k) of neutral and ionic drugs were determined. The results show that the log k measured by the ILCCC based on two liposomes have a good linear fitting (R2 = 0.86). Moreover, the linear relationship between ILCCC system and other related research systems (octanol-water system and immobilized artificial membrane (IAM)) was analyzed, and the results indicate that SPC/PE/Chol ILCCC, DSPC/PE/Chol ILCCC and IAM systems have good fitting results, R2 values are 0.86 and 0.78, respectively. In addition, the normalization coefficients of ILCCC and IAM systems obtained by the linear free energy relationship (LFER) analysis are close and the d value is small. In short, the ILCCC is a simple and feasible method for studying drug membrane permeability.

Development of a retention prediction model in ion-pair reversed-phase HPLC for nucleoside triphosphates used as mRNA vaccine raw materials.

The International Conference on Harmonization guidelines for quality on pharmaceutical development recommends a systematic development approach including robustness studies which assure performance of manufacturing and analytical method development of drug product. It was demonstrated that the retention prediction model for nucleoside triphosphates (NTPs) on ion-pair reversed-phase HPLC was developed by a highly accurate Kawabe's model which supports the development of robust HPLC methods. As NTPs and its derivatives are typically used for Messenger ribonucleic acid (mRNA) vaccine production, adenosine-5'-triphosphate (ATP), guanosine-5'-triphosphate (GTP), cytidine-5'-triphosphate (CTP), 5-methylcytidine-5'-triphosphate (m5-CTP), uridine-5'-triphosphate (UTP), 5-methyluridine-5'-triphosphate (m5-UTP), pseudouridine-5'-triphosphate (Ψ-UTP) and N1-methylpseudouridine-5'-triphosphate (m1Ψ-UTP) were applied for prediction model development. By a comparison of the predicted retention factor in eight studied samples with the retention factor measured under six isocratic conditions, the absolute prediction error was 0.075 and also the prediction error (%) was 2.70%. In practical examples, analytical method for residual ATP, GTP, CTP, and m1Ψ-UTP in the commercial mRNA-based drugs and purity method for UTP derivatives were optimized by QbD approach. The design space for the minimum resolution between adjacent peaks was simulated with the models developed to evaluate the robustness of peak separation, and the optimal mobile phase condition was also simulated. As a conclusion, the desired peak was successfully separated under the optimized condition, and we thought that these retention models could optimize the mobile phase condition of the NTP analysis method for applying to various quality tests, such as quantity, purity and identity test for NTPs and its derivates in the mRNA-based drugs.